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J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.
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Published in final edited form as:

J Thromb Haemost. 2013 June ; 11(Suppl 1): 46–66. doi:10.1111/jth.12253.
Evolutionary origins of the blood vascular system and

endothelium
Rita Monahan-Earley1,2, Ann M. Dvorak1,2, and William C. Aird1,3,4
1TheCenter for Vascular Biology Research, Beth Israel Deaconess Medical Center, Boston, MA
02215
2Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215
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3Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215

4Mount Desert Island Biological Laboratory, Salisbury Cover, ME 04672
Abstract
Every biological trait requires both a proximate and evolutionary explanation. The field of vascular
biology is focused primarily on proximate mechanisms in health and disease. Comparatively little
attention has been given to the evolutionary basis of the cardiovascular system. Here, we employ a
comparative approach to review the phylogenetic history of the blood vascular system and
endothelium. In addition to drawing on the published literature, we provide primary ultrastructural
data related to the lobster, earthworm, amphioxus and hagfish. Existing evidence suggests that the
blood vascular system first appeared in an ancestor of the triploblasts over 600 million years ago,
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as a means to overcome the time-distance constraints of diffusion. The endothelium evolved in an

ancestral vertebrate some 540–510 million years ago to optimize flow dynamics and barrier
function, and/or to localize immune and coagulation functions. Finally, we emphasize that
endothelial heterogeneity evolved as a core feature of the endothelium from the outset, reflecting
its role in meeting the diverse needs of body tissues.
INTRODUCTION
Every biological trait requires both a proximate and evolutionary explanation (reviewed in
[1]). Proximate explanations (“how”) employ traditional tools, including biochemistry,
molecular biology and cell biology to describe the anatomy, physiology and ontogeny of a
trait at the level of a modern day organism. Evolutionary explanations (“when and why”)
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employ a combination of the fossil record, and comparative morphology and DNA
sequences to describe the phylogenetic history of a trait and the fitness advantage that the
trait provides at the level of a population or species [2].
The field of cardiovascular biology is largely concerned with proximate mechanisms. How
does the heart generate force? How do blood vessels form during development? How do
blood vessels and their endothelial lining function in health and how do they become
Contact information: William C. Aird, M.D., Beth Israel Deaconess Medical Center, Molecular and Vascular Medicine; RN-227, 330
Brookline Ave., Boston MA 02215. waird@bidmc.harvard.edu; phone: 617-667-1031; fax: 617-667-1035.
Monahan-Earley et al. Page 2
dysfunctional in disease? By contrast, little attention is paid to the evolutionary mechanisms

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of the cardiovascular system. When and why did the cardiovascular system evolve in the first
place? Why are certain blood circulatory systems open, while others are closed? Why are
some systems lined by endothelium, whereas others have no cell lining? These are important
questions because they provide insights into the design constraints, path dependence, trade-
offs and selective pressures that underlie human physiology and vulnerability to disease.
The goal of this review is to explore the evolutionary origins of the blood vascular system
and endothelium. There is no trace of the cardiovascular system in the fossil record.
Molecular phylogenetic analyses have yielded interesting, though limited insights into
evolutionarily conserved mechanisms of heart development and tube formation. By contrast,
comparative biology provides a rich source of information that can be used to infer and
reconstruct the evolutionary history of the cardiovascular system. We will begin with an
overview of body plans in extant animals. Next, we will discuss concrete examples (and
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introduce primary ultrastructural data) from a spectrum of representative species, with a

particular emphasis on invertebrates. Finally, we will use these data to draw conclusions
about the evolutionary origins of the blood vascular system. A glossary of terms and
concepts used in the current review is included in the online Supplement.
BODY PLANS
All animals have evolved to survive and reproduce, and to do so they share common tasks
that include the capture, ingestion, absorption and distribution of food/nutrients; the
acquisition and distribution of oxygen for cellular respiration; and the excretion of metabolic
wastes and undigested materials [3]. Different species use different strategies to achieve
these goals. Such strategies diverge most prominently with changes in body size and
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complexity. However, the number of identifiable themes or body plans (including the
structural design of cardiovascular systems) is limited by developmental /genetic constraints
and the laws of chemistry and physics. A consideration of these designs provides important
insights into the evolutionary history of the blood vascular system and endothelium.
A word on taxonomy
Eukaryotes consist of two kingdoms, the Protozoa and Animalia (Metazoa). For our
purposes, Metazoa, which represent multicellular eukaryotes, can be further classified in one
of two ways. The first classification employs molecular and/or morphological data to
describe the evolutionary relationships among major metazoan lineages. The results, which
may be represented in the form of phylogenetic trees (an example is shown in Fig. 1), can be
used to infer evolutionary histories. The deep branches on the animal tree of life remain
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controversial. Although the details of the metazoan phylogenic tree continue to be revised as
new evidence emerges, several important themes emerge. The origin of the Metazoa dates
back to approximately 770–850 million years. The most primitive living phylum of animals
is the Porifera (sponges), followed by Cnidaria (corals and jellyfish) and Ctenophora (comb
jellies). Only two germ layers (endoderm and ectoderm) develop in these latter phyla.
Hence, they are called diplobastic animals. Between 600 and 700 million years ago, a new
body plan emerged that demonstrated bilateral symmetry and a third germ layer

(mesoderm). 1 These animals, referred to as triploblasts, gave rise to two separate lineages:
the protostomes and deuterostomes.2 The deuterostome lineage gave rise to the chordates
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(including cephalocordates, urochordates and vertebrates) as well as hemichordates (acorn

worms) and echinoderms (e.g., sea urchins and starfish). Protostomes are further divided
into two groups: the Ecdysozoans (including the arthropods and nematodes) and the
Lophotrochozoans (including the mollusks and annelids).3 As we shall see, these various
branch points mark important transitions in the development of vascular systems.
A second, simpler classification separates Metazoa into vertebrates (those animals that
possess a backbone/vertebral column consisting of bone or cartilage) and invertebrates
(those that do not possess such a structure). Vertebrates, which constitute the subphylum
Vertebra of the phylum Chordata, comprise only 3% of all living animal species.
Invertebrates, which span over 30 phyla, constitute the remainder of metazoan species.
Invertebrates are highly paraphyletic4, and thus display an enormous diversity of body plans.
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By contrast, all vertebrates are derived from a common ancestor, and are thus constructed on
a single common body plan.5 Although the invertebrate-vertebrate dichotomy carries little
taxonomic meaning, it allows for a distinction between the prototypic vertebrate
cardiovascular system and a broad spectrum of vascular phenotypes in invertebrates, many
of which arose independently in response to common selective pressures.
Size matters
Larger body size is a major trend in animal evolution. Changes in size mandate changes in
structural design. All unicellular and multicellular animals depend on diffusion to supply
oxygen and nutrients, and to remove carbon dioxide. Diffusion, while energetically
inexpensive, is a very slow process and works only over small distances (diffusion path < 1
mm).6 A change in body size (whether for a single cell or a multicellular organism)
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disproportionately changes the ratio of surface area to volume. Specifically, as a solid 3-

dimensional body enlarges, its surface area increases in proportion to the radius squared (r2),
whereas its volume increases more rapidly (r3). At some point, the cell will reach a size
where its surface area cannot meet the needs of its volume. Single cells optimize their
surface area-to-volume ratio by developing folded surfaces, or a flattened or thread-like
shape. Another strategy to increase size is to incorporate many cells into a single organism.
Simple multicellular organisms (diplobastic animals and some of the early triploblastic
animals, such as flatworms) obtain oxygen by diffusion alone. They do so by minimizing
1Bilaterality is associated with cephalization (anterior-posterior head-to-tail body axis); a dorsoventral back-to-front axis; and a
division of the body into left and right sides.
2The deuterostomes and protostomes are considered superphyla of Metazoa. They differ primarily in their embryonic origins. For
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example, in protostomes, the blastopore becomes the mouth, whereas in deuterostomes it becomes the anus.
3These two groups were initially proposed based on molecular phylogenetic analyses of the protostomes (particularly similarities in
18S rRNA genes).
4A paraphyletic group is a group whose member species are all descendants of a common ancestry, but that does not contain all the
species descended from that ancestor.
5For example, all vertebrates have an axial skeleton with vertebral column, brain complexity, an anterior mouth, a posterior anus, a
digestive tract, a liver, kidneys, a ventrally located heart (two hearts in the case of hagfish), a closed circulatory system with an
endothelial lining, a neural crest, and acquired immunity.
6According to Fick’s first law, diffusion is proportional to the concentration gradient and surface area and inversely proportional to the
distance. dS/dt=D x A x dC/dx, where dS/dt is the rate of transport, A is the area though which diffusion occurs, dC/dx is the
concentration gradient and D is the diffusion coefficient of the substance. See LaBarbera M. Principles of design of fluid transport
systems in zoology. Science 1990;249: 992–1000.

metabolic demands, by assuming a body geometry that maximizes the surface area, by
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localizing most of their cells at the environment/body interface and/or by pumping external
environmental water to their internal surfaces. However, these strategies have inherent
design constraints that place an upper limit on body size. To achieve further 3-dimensional
increases in size, it is necessary to employ internal transport and exchange systems (i.e.,
circulatory systems) to provide bulk flow delivery of substances (e.g., gases, nutrients,
wastes) to and from each cell in the body.
Circulatory systems
A circulatory system is any system of moving fluids that reduces the functional diffusion
distance that nutrients, gases and metabolic waste products must traverse regardless of its
embryological origin or its design [3]. Circulatory systems are highly varied. In diploblasts,
they involve circulation of seawater into a body cavity that is open to the environment. In
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triploblasts, however, circulatory fluid is an internal, extracellular, aqueous medium

produced by the animal and distributed either through body cavities or through integrated
networks of vessels, sinuses and pumping organs. Circulatory fluids can be moved by ciliary
function or muscle action. Directional flow is achieved by means of coordinated peristaltic
waves of contraction and/or the presence of one-way valves. There are two internal
circulatory systems: coelomic and blood vascular. Most triploblastic animals possess both a
coelomic circulatory system and a blood vascular system.7
Coelomic circulatory systems—Some small and flat triploblastic animals (e.g.,

flatworms) have no system of internal fluid support (i.e., they lack a coelom or blood
vascular system). They are called acoelomates (Fig. 2). The space between their ectoderm
and endoderm tissue layers is filled with a meshwork of mesodermal cells called
parenchyma. These creatures obtain all of their oxygen and food by simple diffusion across
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the skin and gut and throughout the intercellular medium.8 If there are fluid-filled clefts in
this meshwork, the animal is termed a pseudocoelomate.9 However, most triploblastic
animals have a fluid-filled body cavity between the outer body wall (ectoderm) and the
digestive tube (endoderm), termed the coelom. The coelom is lined by mesoderm-derived
epithelium (termed mesothelium), with the apical surface of the mesothelial cells facing
towards the lumen.10 The cavity is filled with coelomic fluid, which in some cases contains
cells (termed coelomocytes). Coelomic cavities have never developed a pumping system.
Instead, fluid transport is produced by cilia on the surface of the mesothelial cells, by
contraction of mesothelial cells (which have acquired a myoepithelial phenotype, and are
thus termed myoepithelial cells), or by contraction of body wall muscle. The coelom is
usually subdivided into multiple compartments by septa and mesenteries (Fig. 2).11 In
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7There are some exceptions such as higher leeches and the nemertines, in which a coelom exists without a blood vascular system.
Conversely, animals with an open circulation (e.g., mollusks and arthropods) have a blood vascular system, but a significantly
regressed coelomic system.
8The largest have highly branched guts that carry out the functional equivalent of internal transport (see Fig. 1). Reliance on the gut
for diffusion of gases and nutrients limits the degree to which the gut may be regionally specialized, e.g., for digestion, absorption.
9The pseudocoelom, also referred to as a blastocoelom, is not formed from mesoderm and is not lined by mesothelium. It may
represent remnants of the embryonic blastocoel. See Brusca R.C., Brusca G.J. Invertebrates. Sunderland, MA: Sinauer Associates,
2003, p. 48–9.
10Mesothelial cells also called myoepithelial cells, peritoneal cells, and cardiac cells.
11Examples in mammals include the pericardial cavity, pleural space, and peritoneal cavity.

addition to providing convective flow of gases, nutrients and wastes, the appearance of the
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coelom allowed organs to move freely, rather than being embedded in solid mesoderm
tissue. The coelom also provided space for organ development. Functionally, a coelom can
absorb shock or provide a hydrostatic skeleton essential for certain types of movement.12
Because they tend to be compartmentalized, coelomic cavities function in the local
circulation of fluid. In contrast, blood vascular systems have evolved in the vast majority of
coelomates to provide bulk fluid transport throughout the body of segmented animals.
Blood vascular systems—The blood vascular system consists of blood-filled spaces

(vessels, sinuses, hemocoels, and/or pumping organs) within the connective tissue
compartment, which is continuous around and between all tissue layers in the body [4]. In
invertebrates, the spaces are lined only by matrix. Vertebrates have evolved a secondary cell
lining, termed endothelium. Fluid transport is produced by contraction of specialized
mesothelial cells (myoepithlial cells) and/or muscular pumps.13 The blood vascular system
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is used for the transport of substances (e.g., nutrients, oxygen, carbon dioxide), hydraulic
force generation (e.g., head-foot protrusion in mollusks and penile erection in vertebrates),
regulation of heat (e.g., via countercurrent flow), ultrafiltration (e.g. in the kidney), defense
(e.g., through delivery of clotting factors, immune factors/cells) and whole-body integration
(e.g., hormonal regulation). Blood vascular systems follow one of two principal designs:
open or closed. In keeping with their paraphyletic origins, invertebrates display diverse
phenotypes ranging from open to closed systems. In contrast, all vertebrates have a closed
cardiovascular system. As we shall discuss, the division between closed and open systems is
not always clear-cut.
Open circulatory systems occur in arthropods (e.g., insects and crustaceans) and non-
cephalopod mollusks (e.g., clams, snails and slugs). In these animals, the mesoderm forms
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coelomic cavities during embryogenesis. However, the cavities and their cell lining regress
in the adult. Some populations of mesodermal cells reaggregate and form the blood vessels
(e.g., the dorsal vessel in insects). The system is considered open because the blood (called
hemolymph) empties from a contractile heart and major supply vessels into the body cavity
(termed a hemocoel), where it directly bathes the organs. In other words, the hemocoel is
bordered not by mesoderm-derived mesothelial cells, but rather by the basal surface of the
tissue cells themselves.14 Thus, once blood empties from the lumen of the distributing
vessels, there is no distinction between hemolymph and interstitial fluid/extracellular fluid.
Hemolymph returns to the heart either through ostia in the ventricle (arthropods) or via the
atrium (mollusks).15 Compared with the closed circulation in lower invertebrates (e.g.,
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12In fact, the coelom may have evolved initially to provide a hydrostatic skeleton for early soft-bodied, bilaterally symmetrical
animals that assumed a crawling or burrowing lifestyle.
13The following types of pumps or hearts may be distinguished: 1) myoepithelial cell-containing peristaltic pulsating vessels which
propel blood by peristalsis (e.g., annelids and amphioxus), 2) cardiomyocyte-containing tubular hearts (which represent an adaptation
of an original peristaltic design) whose beating is synchronous or near-synchronous (e.g., insects); and 3) cardiomyocyte-containing
chambered hearts whose beating is synchronous or near-synchronous (e.g., mollusks and vertebrates). The latter two designs are
controlled myogenically or neurogenically. See Xavier-Neto, et al. Parallel avenues in the evolution of hearts and pumping organs.
Cell Mol Life Sci 2007;64:719–34.
14The hemocoel is not a coelom. It may be viewed as a persistent blastoceolic remnant, which has been secondarily derived during
evolution. See Brusca R.C., Brusca G.J. Invertebrates. Sunderland, MA: Sinauer Associates, 2003, p. 476.
15Mollusks and vertebrates are the only animals that possess a chambered heart with at least one ventricle and atrium. The occurrence
of chambered hearts in these two phylogenetically distant groups is an example of convergence.

annelids), the open circulation boasts a more efficient pump. Whereas annelids rely on
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peristaltic blood vessels to propel blood, animals with an open circulation have evolved true
hearts (containing muscle striations and Z-bands), which display automatic and
synchronized beating. However, compared with more advanced closed systems (e.g., in
cephalopods and vertebrates), open circulatory systems have a larger blood volume and
lower flow rates/pressures. Indeed, the velocity and blood pressure drop abruptly once the
blood leaves the heart and vessels and enters the hemocoel. As a final point of comparison,
open systems provide flow to organs in series, such that tissues lying further downstream of
the heart receive less oxygen compared with more proximal organs. By contrast, the
capillary networks of closed circulatory systems are arranged in parallel allowing for equal
(and regulated) distribution among tissues.
In insects, the open circulation is not responsible for delivering oxygen. Oxygen delivery is
carried out by an elaborate, highly branched tracheal system, which facilitates diffusion to
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each and every cell of the body. As a result, insects have a larger capacity for aerobic
metabolism compared with other with open-circulation invertebrates.
Closed circulatory systems occur in a wide variety of invertebrates including annelids,

cephalopods (e.g., octopus and squid) and non-vertebrate chordates, as well as in
vertebrates. In closed systems, the blood remains inside distinct channels or chambers,
where it is physically separated from the intercellular fluid, body cells and coelom.16 Closed
systems consist of collecting and distributing vessels, usually with a central meeting site in a
propulsive pump. Exchange with the interstitial fluid and body cells takes place in special
areas such as capillary beds or plexi, where the walls are thin to optimize diffusion. The
closed system of invertebrates consists of a network of spaces in the extracellular matrix
between epithelial cells where the coelom contacts coelom (e.g., mesentery or septa) or
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where coelom contacts endoderm or ectoderm (Fig. 2). Thus, the wall of these vessels
consists of basement membranes derived from different kind of epithelia and the vessels are
outlined by the basal surfaces of these cells. In some cases (e.g., annelids), blood is
propelled by the pumping action of the blood vessels, which in turn is mediated by the
contraction of specialized, differentiated coelomic mesothelial cells (myoepithelial cells), or
by muscular blood vessels that contract in peristaltic waves.17. In other cases (e.g., in
cephalopods), chambered hearts have evolved to promote fluid movement.
In vertebrates, the closed vascular system consists of a series of closed vessels with an
endothelial lining, invested with smooth muscle cells or pericytes. Vertebrate blood vessels,
16Some authors have used “closed” to describe systems lined by endothelium (e.g., see McMahon B.R.. Comparative evolution and
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design in non-vertebrate cardiovascular systems. In D. Sedmera and T. Wang (Eds.), Ontogeny and phylogeny of the vertebrate heart.
New York, NY: Springer, 2012; Shigei T., Tsuru H., Ishikawa N. and Yoshioka K. Absence of endothelium in invertebrate blood
vessels: significance of endothelium and sympathetic nerve/medial smooth muscle in the vertebrate vascular system. Jpn J Pharmacol
2001;87:253–60). Our definition does not require the presence of a cell lining.
17These latter cells, which contain contractile myofibrils, may remain in the coelomic cavity or they may delaminate and constitute a
differentiated contractile cell layer between the hemal cavity and coelomic epithelium (giving rise to a multilayered blood vessel wall).
See Munoz-Chapuli R., Carmona R., Guadix J.A., Macias D., Perez-Pomares J.M. The origin of the endothelial cells: an evo-devo
approach for the invertebrate/vertebrate transition of the circulatory system. Evol Dev 2005;7:351–8; Perez-Pomares JM, Gonzalez-
Rosa JM, Munoz-Chapuli R. Building the vertebrate heart – an evolutionary approach to cardiac development. Int J Dev Biol
2009;53:1427–43. In addition to giving rise to contractile myoepithelial cells, coelomic epithelial cells are believed to differentiate into
hemocytes during development (see Hartenstein V., Mandal L. The blood/vascular system in a phylogenetic perspective. Bioessays
2006;28:1203–10). Hemocytes are diverse in structure and function, but participate in functions similar to counterparts in vertebrates.

while contractile, do not propel blood. Rather, transport is mediated by a central muscular
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pump. Most vertebrates have a lymphatic system, which collects and recycles interstitial
fluid back to the circulation. There are variations on the common vertebrate plan, many of
which are related to different requirements of living in water or on land (reviewed in [5, 6]).
For example, fish have a single circulatory system, consisting of an undivided heart with a
single atrium and single ventricle in series with oxygen-exchanging gills. By contrast, adult
birds and mammals have a double circulatory system in which the heart is completely
divided into right and left sides, resulting in separation of deoxygenated and oxygenated
blood. The circulatory systems of lungfish, amphibians and reptiles demonstrate a broad
array of intermediate designs, each of which is characterized by partial separation of the air
breathing organ and the systemic circulation, and thus the potential for left-right and right-
left shunts.
The idea that cardiovascular systems are either closed or open is an oversimplification [7].
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For example, in many mollusks and crustaceans, blood is ejected from the heart into a highly
ramified network of branching vessels before emptying into the hemocoel. This is illustrated
in the lobster (see section below), and even more prominently in the sea snail, Haliotis,
where the abdominal arteries end up in a complex meshwork of small capillary-like vessels
[8].18 Moreover, studies in drosophila have shown that blood flows along preferred routes in
the open hemocoel despite the absence of discrete return channels [9]. Conversely, the
closed system of vertebrates contains vascular beds, such as the sinusoids of the liver, spleen
and bone marrow, where there is direct contact between blood and the interstitial space. In
hemochorial placentation (e.g., in primates), the maternal spiral arteries become open ended,
and blood is released into a placental labyrinth where it bathes the chorionic villi and is
drained by the spiral veins. Such an arrangement is highly reminiscent of an open system.
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Endothelium
Invertebrate vessels are always lined by extracellular matrix. Only vertebrates possess a true
endothelial lining, defined as a layer of epithelial cells expressing basoapical polarity (with
the apical surface facing the lumen), intercellular junctions and anchoring to basement
membrane. The blood vessels of some invertebrates, including cephalopods, annelids, and
amphioxus (discussed next) have cells clinging to the luminal surface, internal to the
basement membrane. These cells have sometimes been referred to as “endothelial cells”
[10–13]. However, they form an incomplete lining, they lack intercellular junctions typical
of vertebrate endothelial cells and they rarely appear attached to the underlying basal lamina.
A more appropriate term for this cell type is an amoebocyte. It likely represents a type of
circulating hemocyte, which may or not be an evolutionary precursor of the endothelial cell.
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Gas exchange
The circulatory system has co-evolved with gas exchange mechanisms. Some invertebrates
obtain their oxygen by simple diffusion through the skin. Integumentary gas exchange is
characteristic of small soft-bodied animals with high surface:volume ratios. Gas exchange in
18There is no structural equivalent of a vertebrate artery in invertebrates. The term “artery” is often used to describe distributing
vessels that supply blood downstream of a pump. In other words, an artery is defined by its efferent relationship to the pump and not
by its wall structure.

the majority of marine and many freshwater invertebrates occurs via gills. Terrestrial
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invertebrates employ specialized invaginated structures, including book lungs (spiders) or

trachea (insects). Many invertebrates have metal ion-containing respiratory pigments to
increase the oxygen carrying capacity of their blood. For example, hemoglobin is found in
many annelids, as well as some crustaceans, insects and mollusks, whereas hemocyanins are
the most commonly occurring respiratory pigments in mollusks and arthropods. Respiratory
pigments are usually carried in solution in blood, but they are occasionally packaged in
blood cells. Vertebrates breathe through their gills, skin and/or lungs. All vertebrates with
the exception of Antarctic icefishes (Channichthyidae) have hemoglobin and red blood cells.
CASE STUDIES
In this section, we will consider the blood vascular systems of four different animals: 3
invertebrates and one basal vertebrate. These examples will be used to highlight features of
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cardiovascular design in the animal kingdom. We begin with an example of an open

circulation (lobster). We then turn to three examples of a closed circulation (earthworm,
amphioxus and hagfish). The reader is referred to the online Supplement for a description of
the methods employed and for additional figures.
Case study 1: The lobster (Homarus americanus)

Lobsters belong to phylum Arthropoda, subphylum Crustacea. Like other arthropods, they
have an open circulation (Fig. 3A, Supplemental Fig. I). They have a well-developed single-
chambered heart suspended in a pericardial sinus in the dorsal thorax by several pairs of
alary ligaments (Fig. 3B). These elastic ligaments are stretched during systole. In diastole,
they expand the heart, which results in passive filling with hemolymph from the pericardial
sinus through three pairs of muscular valved ostia [14]. The heart is comprised of striated
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cardiac muscle cells and is conspicuous for its absence of an endocardial lining (Fig. 3C,
Supplemental Fig. II). The heart empties into 7 arteries, which then branch multiple times
along their length into thin-walled microvessels before emptying into tissue lacunae (i.e., the
hemocoel) (Figs. 3A, 3F) [15]. Muscular cardio-arterial valves are present at the origins of
all but the dorsal abdominal artery. Blood is collected into a series of interconnected sinuses
and passes through either the gills or the branchiostegal sinus before being delivered back to
the pericardial sinus surrounding the heart [15].
Arteries leaving the heart are trilaminar vessels consisting of an inner acellular tunic interna,
a tunica intermedia which contains fibroblast-like cells, and an outer tunica externa (Fig. 3D,
Supplemental Fig. III) [16, 17]. Of the 7 arteries, only the dorsal abdominal artery has
occasional muscle fibers in its wall [16, 18]. Electron microscopy reveals the presence of an
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internal lamina containing microfibrils and a medial lamina composed of a dense network of
microfibrils (Fig. 3E, Supplemental Fig. IV) [19]. All invertebrates lack elastin. Nonetheless,
lobster arteries demonstrate non-linear elasticity (typical of vertebrate arteries) [16]. This
property my be related to the reorientation of the microfibrils within the vessel wall [20].
Previous studies in lobster have demonstrated heart rates of 61–83 beats per minute [16, 21],
and systolic pressures of approximately 20 mmHg [16]. Although all but one of the arteries
lack muscle, they contract with exposure to cardioactive drugs, suggesting that they are not

simply passive supply tubes but rather are used to regulate arterial resistance.[16] Oxygen
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exchange in lobsters takes place in gills. Oxygen transport is facilitated by the copper-
containing respiratory pigment, hemocyanin [22], which is found in solution in the
hemolymph. Lobster blood contains hemocytes (Fig. 3C, Supplemental Fig. II). Between 3
and 11 morphologically distinct types of hemocytes have been described [23]. These cells
are likely involved in innate immunity and coagulation.
In summary, the lobster has evolved highly efficient cardiovascular and respiratory systems
to meet its metabolic requirements. Indeed, the very fact that mollusks and arthropods
represent the largest and most diverse phyla in the animal kingdom is testament to the
enormous success of the open circulatory design.
Case study 2: The earthworm (Lumbricus terrestris)

The earthworm belongs to the phylum, Annelida. Like most triploblastic animals, the
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earthworm has a well-developed coelom and a blood vascular system. The coelom is highly
compartmentalized, with distinct left and right cavities housed in each body segment. The
coelomic fluid provides an important hydrostatic skeleton that enables locomotion.19 The
blood vascular system is closed. There is no heart. Rather, circulation is carried out by
contractile blood vessels. There are three main longitudinal vessels: the dorsal vessel, the
ventral vessel and the subneural vessel (Fig. 4A, Supplemental Fig. V). The largest and most
conspicuous vessel in the earthworm is the dorsal vessel, which lies on the dorsal surface of
the alimentary tract and traverses the length of the animal (Fig. 4B, Supplemental Fig. VI)
[24]. The dorsal vessel collects blood from other vessels and drives it forwards through
contractile peristaltic waves that originate at the posterior end of the animal and move
forward [24, 25]. Anteriorly the dorsal vessel ceases to be a collecting vessel and gives rise
to five pairs of large commissures that connect to the ventral vessel. These commissures
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contain lateral “hearts” (pseudohearts), whose contractions are asynchronous with those of
the dorsal vessel. The ventral vessel, which is suspended in the mesentery beneath the gut, is
the main distributing channel, sending blood forwards anterior to the hearts, and posteriorly
behind the hearts. The ventral vessel has no peristaltic activity. In each segment the ventral
vessel gives rise to segmental vessels and capillaries (Fig. 4C and Supplemental Fig. VII
show capillaries in the body wall), through which blood is carried back to the dorsal vessel.
Blood also flows posteriorly through the subneural vessel, which lies ventral to the nerve
cord.
A previous study in L. terrestris demonstrated a resting dorsal vessel pulse rate of 11 +/− 2.1
beats per minute [24]. The giant earthworm (Glossoscolex giganteus) reaches sizes of 500–
600 g (the body weight of a typical earthworm used in our studies is 3–4 g) and may be as
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long as 120 cm. Frequency of peristalsis in the dorsal vessel of the giant earthworm was
measured as 8 per minute, with an average systolic and diastolic pressure of 24 and 14 cm
H2O, respectively, in resting healthy specimens [25]. Pressures in the ventral vessel in
response to lateral heart contractions can exceed 100 cm Hg [25]. There is evidence that
blood flow is physiologically regulated in annelids. For example, dorsal vessel pulse rates
19The action of the body wall muscles on the coelomic fluids provides the hydraulic changes associated with locomotion.

are increased in L. terrestris after forced exercise [24]. Moreover, the load or degree of
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filling of the dorsal vessel determines the frequency as well as the force of contraction of the
peristaltic waves [24, 25]. Finally, a previous study in L. variegatus demonstrated a role for
biogenic amines in regulating the dorsal vessel pulse rate [26].
In electron microscopy, blood vessels are readily identified by the presence of electron-dense
hemoglobin particles within the lumen (Fig. 4D, Supplemental Fig. VIII). The vessels are
outlined by the basal lamina/basement membrane of mesodermal cells, mesodermal and
endodermal cells (of the gut), or mesodermal and ectodermal cells (of the skin). The
thickness of this extracellular layer (basement membrane) varies across the vascular tree
[27]. Mesodermal cells have varying numbers of microfilaments (Fig. 4E, Supplemental Fig.
IX) [27, 28]. Microfilament-rich mesodermal cells (myoepithelial cells) are presumed to
have a contractile function. Scattered amoebocytes are found on the inner surface of the
lumen (Fig. 4F, Supplemental Fig. X) (these cells can also be seen on one-micron Giemsa-
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stained sections of blood vessels, as shown in Supplemental Fig. XI) [27].20 The
amoebocytes contain electron-dense granules and vesicles [27]. In tracer experiments in
which colloidal gold or horseradish peroxidase was injected into the blood vessels, most of
the vesicles in the amoebocytes were filled with tracer, suggesting that they are endosomes
[27]. Amoebocytes are distinct from endothelial cells. They do not appear to be firmly
attached to the basement membrane. They cover only a small fraction of the surface of the
blood vessel. There is no evidence for junctional complexes between neighboring cells.
Finally, the large number of dense granules is atypical for endothelial cells. In summary,
blood vessels of the earthworm are lined by basement membrane and lack an endothelial
layer.
Earthworms have no specialized respiratory organs.21 Rather, gas exchange takes place
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across the general body surface. The earthworm has an extensive intradermal capillary
network, which provides a high surface area for such exchange. The blood contains a high
molecular weight (3.6 MDa) hemoglobin dissolved in the plasma [29, 30].22 The oxygen
content in blood samples from the dorsal vessel of giant earthworms has been reported to be
between 0.7 and 9.8% [31].
In summary, the earthworm provides an example of an invertebrate with a closed blood

circulatory system. Unlike its vertebrate counterpart, the closed circulation of annelids lacks
a true heart and consists of blood vessels that are delimited by the basal surface of epithelial
cells, some of which exhibit a contractile function.
Case study 3: Amphioxus (Branchiostoma lanceolatum)

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Amphioxus belongs to the phylum Chordata, subphylum Cephalochordata, which is the

most basal subphylum of the chordates [32, 33].23 They have extensive coelomic cavities
20These cells have been variously referred to as vasothelial myobasts, amoebocytes, blood cells, hemocytes or endothelial cells. See
Hama, K. The fine structure of some blood vessels of the earthworm, Eisenia foetida. J Biophys Biochem Cytol 1960;7:717–24 (and
references therein).
21In fact, the giant earthworm represents the largest living terrestrial animal without specialized respiratory organs.
22A recent study demonstrated the use of ultra-pure earthworm Hb as a means to improve oxygen delivery in hamsters with severe
anemia. See Elmer J, Palmer AF, Cabrales P. Oxygen delivery during extreme anemia with ultra-pure earthworm hemoglobin. Life Sci
2012;91:852–9.

and a closed circulation. The blood does not contain circulating cells or hemoglobin (or any
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other respiratory pigment). As a result, the blood vessels are not readily identifiable.
However, previous tracer studies have yielded a roadmap of the circulatory system in
amphioxus (Fig. 5A, Supplemental Fig. XII) [12, 34, 35]. Amphioxus do not have a heart.
Instead, circulation is accomplished by contractile vessels, including the endostylar artery,
subintestinal vein and hepatic vein. As in vertebrates, blood moves forward ventrally and
backwards dorsally. The endostylar artery (also called the ventral aorta) is ventral and carries
blood in a cranial direction. The endostylar artery sends vessels to the gills, which comprise
more than 80 pairs of gill slits with adjacent slits separated by gill bars (Fig. 5B,
Supplemental Figs. XIII–XV) [36]. The gills are used for filter feeding, not for oxygen
exchange [37]. The gill bars are of two types: primary and secondary. The primary gill bars
have three longitudinal vessels: the visceral, the skeletal and the coelomic. The secondary
only have two such vessels: the visceral, and skeletal. Blood from the gills moves to the
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dorsal aorta. In the anterior region of the pharynx, the dorsal aorta is paired and lies
immediately under the notochord. It is fused posteriorly. The dorsal aorta sends branches
(segmental dorsal and ventral arteries) to supply the myomeres. It also sends short
mesenteric arteries, which open directly into the plexus of vessels surrounding the intestine
[34]. Blood in the intestinal plexus is drained into the subintestinal vein followed in turn by
the afferent liver vessel, the liver plexus and the efferent liver vessel (also called the hepatic
vein). The hepatic vein runs caudally along the dorsal surface of the liver (a simple blind
diverticulum of the intestine) to ultimately join the common cardinal vein at the sinus
venosus to form the endostylar artery (Supplemental Figs. XV–XVI). Previous tracer studies
have shown that the various afferent and efferent vessels of amphioxus are connected by
extensive plexi of tiny vessels [34].
Blood vessels in amphioxus are not lined by endothelium, but rather by the basal surfaces of
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epithelial cells and extracellular matrix. For example, the dorsal aorta is delimited by the
basal surface of coelomic cells and by connective tissue around the sheath of the notochord
and the hyperpharyngeal groove (Figs. 5B–5C, Supplemental Figs. XVII). Blood vessels in
the gills are lined by varying types of epithelial cells, including coelomic cells, atrial cells,
lateral ciliated cells, lateral pharyngeal cells and medial pharyngeal cells. In the gut, the
blood vessel lumen forms between the basal surfaces of coelomic cells and intestinal
epithelial cells. A previous tracer study demonstrated that the basal lamina in the visceral
vessel of the gill is more permeable to horseradish peroxidase compared with endostylar
artery [13], suggesting vascular bed-specific differences in permeability properties.
In contractile vessels (e.g., the hepatic vein and endostylar artery), the surrounding
mesoderm consists of myoepithelial cells. These cells contain contractile filaments in the
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basal part, next to the basal lamina [12]. Many filaments end in patches of higher density
(Fig. 5D, Supplemental Fig. XVIII). Isolated masses of connective tissue are distributed
around the luminal margin of the hepatic vein near attachment of the vein to the cecum.
23Like vertebrates, they have a hollow dorsal nerve cord, a notochord, segmented muscles, and pharyngeal gill slits. In contrast to
vertebrates, the notochord persists in adults and it is the only skeletal structure in its body. Like other chordates, they possess a series
of V-shaped muscle blocks (myomeres or myotomes). Although fish-like shape, they lack eyes, paired fins, ears, jaws. They are
always half buried in benthic substrate, and filter feed on small particles of plankton.

These finger-like extensions gain entrance into the lumen and form a web-like supporting
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structure for the vessel wall [12].
As in the earthworm, scattered amoebocytes are found clinging to the inner surfaces of
amphioxus blood vessels (Figs. 5E–5F, Supplemental Figs. XIX–XX). These cells, which
are rounded or flattened, are rarely in contact with one another and when they are, there are
no junctional specializations. The cells contain a well-formed Golgi apparatus. They are
packed with polyribosomes and rough endoplasmic reticulum. They also possess many
vesicles. In previous tracer studies, ferritin and horseradish peroxidase were taken up by
coated and smooth surfaced vesicles within the amoebocytes, suggesting that these latter
cells possess endocytotic capability [13]. The most conspicuous finding (which is not
observed in the earthworm amoebocyte) is the presence of tubules in membrane-bound
organelles (granules), which occur at varying orientations (Figs 5G, Supplemental Fig.
XXI). The size of the tubules appears larger than what is observed in Weibel-Palade bodies
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and microtubules in the cytoskeleton. It has been proposed that these tubules are precursors
of cytoplasmic microtubules involved in cell motility, and that the amoebocytes function by
“policing” the luminal surface of the boundary layer, removing residues of filtration and
facilitating exchange of material between blood and surrounding tissues [12]. Others have
suggested that these cells are responsible for depositing and/or clearing the lumen of
extracellular matrix as blood vessels develop in the amphioxus larvae [38, 39].
In summary, despite its close evolutionary relationship with vertebrates, amphioxus seem to
possess a relatively “primitive” closed circulatory system, conspicuous for its absence of a
central heart and endothelial lining, as well as lack of circulating blood cells and respiratory
pigment.
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Hagfish (Myxine glutinosa)

Modern vertebrates are classified into two major groups: the gnathostomes (jawed
vertebrates) and the agnathans (jawless vertebrates). The agnathans are further classified into
two groups, myxinoids (hagfish) and petromyzonids (lamprey). Most evidence suggests that
the hagfish diverged before lamprey and is thus the oldest living vertebrate. Shared traits in
hagfish and jawed vertebrates indicate that the trait was present in the common ancestor of
all craniates.
Hagfish have a closed circulation (Fig. 6A, Supplemental Fig. XXII). Similar to the case in
other fishes, the branchial (i.e., gill) circulation is found in series with the systemic
circulation. The gills are unique among vertebrates in that they are internalized and
organized as pouches, typically 6 on each side in M. glutinosa (Fig. 6B). The hagfish
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circulation also possesses a series of blood sinuses, which are in direct communication with
systemic vessels [40]. The most prominent of these is the large subcutaneous vascular sinus
located between skeletal muscles and the skin, stretching from the tentacles of the snout to
the caudal fin fold. Hagfish can hold up to 30% of their blood volume within the sinus
system [40]. It is believed that the caudal and cardinal “hearts” (composed of skeletal
muscle) function to reintroduce sinus blood into the systemic circulation. The hagfish heart
exhibits a Frank-Starling mechanism [41]. Thus, blood that is redistributed from sinus to
central compartments may serve to increase preload and cardiac output.

Hagfish maintain the lowest arterial blood pressures (dorsal aorta blood pressure 5.8–9.8
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mm Hg) and highest relative blood volumes (18%) of any vertebrate [42–44]. Cardiac output
in M. glutinosa is 8–9 mL min−1 kg−1 [45], which approaches the values seen in some
teleosts. Similar to other fish, hagfish have a double-chambered heart containing a single
atrium and a single ventricle. The heart consists of an avascular mesh of muscle cells (a so-
called spongy heart). There is no coronary circulation. Rather, deoxygenated blood in the
ventricle circulates through spaces (sinusoidal channels or lacunae) in the myocardial wall.
Unlike other vertebrates, the hagfish heart lacks cardioregulatory nerves. The heart rate
ranges from 20–30 beats per minute [45]. Hagfish possess a second cardiomyocyte-
containing chambered heart, the portal heart, which beats asynchronously with the systemic
heart, as it propels blood from the gut to the liver via the common portal vein [45].
We and others have previously shown that hagfish blood vessels are lined by a single layer
of endothelial cells [46–48]. All endothelial cells have vesicles along both the blood and
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tissue fronts of the cell. The lateral borders of endothelial cells are typical for those seen in
mammals. Occasional Weibel-Palade bodies are observed, suggesting that hagfish express
von Willebrand factor. Similar to other vertebrates, endothelial phenotypes vary from one
vascular bed to another. For example, endothelial cells lining the heart sinuses are
attenuated, electron lucent, and contain many vesicular structures (Fig. 6C, Supplemental
Fig. XXIII). Dermal microvessels display a continuous endothelium, with lateral plasma
membrane borders containing specializations typical of other vertebrates (Fig. 6D,
Supplemental Fig. XXIV). The kidney glomerulus contains endothelial cells with few
fenestrae, and many vesicle and vacuoles of varying sizes (Fig. 6E, Supplemental Fig.
XXV). As a final example, ultrastructural studies of the liver demonstrate a discontinuous
endothelium with many gaps (Fig. 6F, Supplemental Fig. XXVI).
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In addition to vascular bed-specific differences in ultrastructure, the endothelium of hagfish

also demonstrates molecular and functional heterogeneity. For example, lectin staining of
various tissues revealed significant differences in lectin binding to the endothelium [46].
Moreover, in intravital microscopy studies of the dermal microvasculature, histamine was
shown to induce neutrophil adhesion in capillaries and postcapillary venules, but not
arterioles [46]. As a final example, arteries from the mesentery and skeletal muscle
demonstrated site-specific mechanisms of endothelium-dependent vasomotor relaxation
[49].
In summary, our findings in hagfish confirm that the closed system of even the most basal
vertebrate is lined by endothelium and that phenotypic heterogeneity of the endothelium is a
conserved property of this cell type.
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EVLOTUTIONARY IMPLICATIONS
When approaching the evolutionary origins of the blood vascular system and the
endothelium, we must consider three important questions. First, when did these systems
evolve? Second, why did they evolve? In other words, what survival advantage do these
structures confer at the level of species? Finally, how did the blood vessels and their

endothelial linings develop ontogenetically (as evolutionary novelties) in the first place? In
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this section, we will consider each question in turn.
When did the blood vascular system and endothelium first evolve?
As we survey the present landscape of body plans, we find a wide variety of blood vascular
systems (Fig. 7). Some are closed, others are open. Some use hearts to propel blood,
whereas others employ pulsatile blood vessels. Only a minority are lined by endothelium.
When and how did all of these diverse structures evolve? The answer is likely through a
combination of homology and convergence.24 The last common ancestor of vertebrates and
annelids, or of vertebrates and mollusks was the ancestor of the protostome-deuterostome
ancestor, which lived between 600 and 700 million years ago [50]. Although the fossil
record is scarce, it is widely believed that this precursor animal was a segmented bilaterian
(triploblastic coelomate) [7]. If we are to accept that the blood vascular system evolved as a
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means to bypass the bulkheads of a segmented animal (see next section), then the first such
system likely arose during this time. Flow would have been mediated by peristaltic vessels,
perhaps like those described in the annelid. Blood probably percolated through spaces in the
extracellular matrix, and thus the system was by definition closed, albeit primitive. This
scenario supports homology of all blood vascular systems. Over the past 600–700 million
years of evolution, the blood vascular system has undergone significant modifications, in
response to selective pressures experienced by individual phyla. In some cases, the blood
vascular system has regressed (e.g., in flatworms and nematodes). In other cases, the
primitive system transitioned to an open circulation. The open system reverted back to a
closed design in an ancestral cephalopod. This is an example of convergent evolution,
whereby an analogous structure (the closed circulation in cephalopods) arose independently
of the closed circulation in other invertebrates (e.g., worms) and in vertebrates. Finally, the
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endothelium is present in all vertebrates, yet distinctly absent in invertebrates. Although

cells appear to cling to the inner surface of some invertebrate closed vessels, these lack the
classical morphological features of endothelial cells. Therefore, the endothelium appeared in
an ancestral vertebrate following the divergence from the urochordates and cephalochordates
between 540 and 510 million years ago. Our findings in hagfish indicate that endothelial
heterogeneity appeared during the same narrow window of time [46].
Why did the blood vascular system and endothelium evolve?

Circulatory systems most certainly evolved to overcome the time and distance constraints of
diffusion, thus permitting increased body size and metabolic rates, as well as increased
levels of integration and organization in Metazoa. Although the primitive coelom provided
bulk flow delivery of substances, these structures never developed an efficient pumping
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mechanism. More importantly, they became increasingly localized to distinct compartments

in the body. Ruppert and Carle hypothesized that the blood vascular system evolved as a
means to provide bulk fluid transport along the entire length of a segmented animal, in effect
bypassing the septal bulkheads [4]. The earliest design may have involved the formation of
spaces in between the basal laminae of endodermal and coelomic epithelia, allowing for
24The reader is referred to an excellent discussion of these concepts in Xavier-Neto, J., et al. Parallel avenues in the evolution of
hearts and pumping organs. Cell Mol Life Sci 2007;64:719–34.

improved distribution of nutrients absorbed by the intestine [51]. Myoepithelial

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differentiation of the visceral layer of coelomic epithelium would have provided the
necessary pumping action to drive nutrients towards the viscera [52]. Subsequent
vascularization of the skin would have facilitated gas exchange at the body surface.
The movement of fluid in blood vascular systems was originally mediated by the peristaltic
motion of certain blood vessels, similar to what occurs in the earthworm and amphioxus.
However, peristaltic pumps lack effective coordination between the fluid that is entering the
contractile region and the fluid that is leaving it. Despite some tweaks to the peristaltic
design, such as the evolution of one-way valves and partial coordination in contractions, the
loss of fluid energy associated with backflow, distension of wall segments ahead of the
stream, and pump reversals would have constrained body size and metabolic activity [53].
This would have provided a selective pressure for the appearance of true hearts, in which
inflow and outflow are tightly coupled via multiple chambers, efficient electrical connection
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between myocytes (hence, simultaneous contraction) and one-way valves [53].
If the closed circulatory system was the ancestral condition, then why did the open
circulatory system evolve in the precursors of mollusks and arthropods? One explanation for
this change in the circulatory blueprint is that the rigid exoskeleton of these animals, which
evolved for protection against predation and physical injury, eliminated the need for a
coelomic hydrostatic skeleton [4]. The subsequent regression of the coelom removed
barriers to bulk transport and therefore the need for a closed system of vessels. At the same
time, the exoskeleton impaired any contribution of body wall musculature to the movement
of blood. This may have provided selective pressure for the evolution of a well-developed
muscular heart, which is a characteristic feature of most animals with an open circulation.25
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Cephalopods are monophyletic with other mollusks, yet they have secondarily evolved a
closed circulation. Moreover, they have a far more efficient heart compared with other
members of the phylum [8, 52].26 It seems likely that these modifications were selected for
to enable an active lifestyle, which includes predatory behavior, swimming and jet
propulsion. The same association between closed circulatory system, highly efficient hearts
and active lifestyles is seen in the vertebrates. There are several reasons why a closed system
might better serve the needs of a highly active animal. First, it provides for a more efficient
(i.e., fractal-like) distribution system, which can be tailored to the unique architecture of the
various tissues and organs.27 Second, closed circulatory systems can be subject to regulation
so that flow is directed preferentially to one organ or another. Third, the closed system
enables carriers to remain inside the vessels and release their cargo where needed. This
allows for homeostatic integration of diverse tissues in the body. Finally, as alluded to
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25This is a nice example of an evolutionary trade-off. In this case, the exoskeleton evolved to provide protection from predators and
physical support. However, body size could no longer increase (hence the evolution of molting), the body wall muscles could no
longer contribute to blood flow (hence the evolution of the heart) and it was no longer possible to breathe through the skin (hence the
evolution of gills).
26The cephalopod heart typically consists of a ventricle and two atria. The myocardium contains multiple layers of striated muscle
cells, which in turn are covered by a coelomic epithelium.
27It is often said that the closed system “allowed” for higher pressures. However, in reality the closed system required high pressures
to overcome the systemic resistance associated with a complex distribution system.

earlier, the closed system provides for parallel flow (equal opportunity) to all organs of the
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body.
The transition from aquatic to terrestrial life required modifications of several body systems,
including those involved with respiration, feeding, locomotion, water balance, and
reproduction. The formation of invaginated gas exchangers (i.e., lungs) reflected the need to
minimize water loss across the respiratory surface [6].28 Ancillary skin breathing (i.e.,
cutaneous gas exchange) was possible so long as the epidermis was highly vascularized and
thin. Animals with thin skin have high evaporative loss. Therefore, skin breathing is
confined to aquatic or semi-aquatic tetrapods, particularly amphibians. Reptiles, which
evolved thick impermeable skin to prevent water loss and provide physical protection, were
the first vertebrates to rely entirely on lungs for gas exchange. A limitation of the single
circulation in fish is that gills receive a greater pressure head than the systemic circulation.
As higher pressures evolved in response to increased metabolic demands and gravitation,
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there was a need to depressurize the gas exchanger. This was accomplished by the separation
of systemic and pulmonary circulations.
In water, buoyancy provides virtual weightlessness, whereas on land, animals are subjected
to gravitational force. Since the density of water and blood are similar, gradients of
hydrostatic pressure in vertical blood columns are counter-balanced by pressure gradients in
surrounding water [54]. Thus, transmural pressure throughout the vasculature of aquatic
animals remains relatively constant. In contrast, on land, gravity causes increased pressure in
the lower part of a fluid column, resulting in increased transmural pressure, pooling of blood
in dependent vasculature, reduced venous return and decreased arterial pressures. This effect
is particularly important in tall animals or in animals with postural behavior (e.g., arboreal or
climbing snakes). Countermeasures to gravitational stress have evolved in tetrapods,
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including higher arterial pressure, baroreceptor reflex, and low compliance tissues (e.g., stiff
skin) [54].
What selective forces were responsible for the appearance of the endothelium in the
ancestral vertebrate? One suggestion is that that the smooth lining provided by the
endothelium minimizes energy required to move blood.29 The endothelium mediates
vasomotor control in the earliest extant vertebrate. Thus, the appearance of the endothelium
may have provided a critical new layer of vasoregulation, allowing for more highly
pressurized systems. Also, the barrier function provided by the endothelium would have
prevented the loss of plasma proteins necessary to maintain equilibrium between osmotic
and hydrostatic pressures [4]. Higher pressures required not only higher osmotic pressures,
but also the formation of the lymphatic system to drain net surplus of interstitial fluid. The
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ancestral vertebrate also evolved novel immune and coagulation mechanisms. Thus, the co-
28The original selection pressure for lungs was aquatic hypoxia, resulting in the appearance of air-breathing fish lungfish. In fish, the
heart receives (and is therefore perfused by) deoxygenated mixed venous blood. It has been hypothesized that the lung (in conjunction
with the coronary circulation) evolved as an adaptation to myocardial hypoxia. See Farmer CG. Evolution of the vertebrate cardio-
pulmonary system. Annu Rev Physiol. 1999;61:573–92. Second,
29Laminar flow is required to minimize the energy needed to move blood through these complex vascular systems. Laminar flow
through a cylindrical tube can be predicted based on vessel diameter, mean blood velocity, and blood density and viscosity
(Reynolds’s number). However, if there are sudden variations in vessel diameter or irregularities in the vessels walls turbulent flow can
result. In turbulent flow a significantly greater pressure is required to move a fluid through the vessels as compared to laminar flow.

evolution of the endothelium during this narrow window of time may be explained in part by
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the role of endothelial cells in localizing these activities to areas of need [55]. Endothelial
cells have been shown to play a pivotal role in the early development of organs, such as the
pancreas and liver [56]. Such bidirectional dialogue between the endothelium and other
tissues may have been instrumental in shaping the evolution of this cell layer.
The fitness advantage of endothelial cell heterogeneity may be explained on several levels
[57]. First, phenotypic heterogeneity reflects the remarkably pleiotropic role of the
endothelium in meeting the varying needs of diverse tissue types. Second, some site-specific
properties reflect a local self-preserving adaptation of the endothelial cells to their particular
microenvironment. Third, phenotypic plasticity provides the endothelium with flexibility to
temporally adjust to a wide range of physiological and pathophysiological influences,
including pathogens and toxins.
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How did the blood vascular system and endothelium evolve?

The coelomic and blood vascular systems (as well as excretory systems) arose within the
mesoderm of triploblastic animals. Indeed, the appearance of the mesoderm provided new
building material for animal construction and allowed for the evolution of increasingly
complex and large animals. At some early evolutionary stage, perhaps in the ancestral
triploblastic bilaterian condition, a subpopulation of mesodermal cells differentiated into a
mesothelium whose apical side faced the coelom and whose basal side faced clefts (i.e.,
blood vessels) between the mesothelial walls [58]. Some of these mesothelial cells acquired
myofilaments and the capacity to contract [55, 58]. If the earliest blood vessels formed
between these myoepithelial cells and gut epithelial cells, then there must have been an
intimate coordination between mesodermal and endodermal lineages. In vertebrates, the
mesothelium no longer contributes to the vasculature. Instead, endothelial cells are derived
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from distinct mesodermal zones arising from the primitive streak, including the lateral plate
mesoderm, cardiac mesoderm and paraxial mesoderm. Other components of the vascular
wall, including smooth muscle cells, are derived from mesoderm or the neural crest (an
ectoderm derivative found exclusively in vertebrates). Thus, at some point following the
divergence of the cephalocordates, the mesoderm of the ancestral vertebrate acquired novel
specialization, which allowed for the formation of endothelium.
There is an interesting body of literature debating the degree to which the drosophila and
vertebrate hearts are homologous [53, 58–60]. An important concept is that homology can
apply to different levels of organization (e.g., genetic, molecular, cellular, tissue structure,
function) [3, 53].30 For example, the drosophila and vertebrate hearts are hardly
homologous in three-dimensional structure (at least in the adult stages). However, the
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drosophila and vertebrates share orthologous genes involved in making a heart, including
tinman/Nkx2-5. Indeed, it has been argued that the homologies lie at the level of gene
regulatory pathways that make a muscle cell rather than at the level of the organ (i.e., the
30Brusca offers a nice example of the importance of the level of analysis being considered: The wings of bats and birds are
homologous as tetrapod forelimbs, but they are not homologous as “wings” because wings evolved independently in these two groups
(i.e., the wings of bats and birds do not share a common ancestral wing). See Brusca, R.C. & Brusca, G.J. Invertebrates, (Sinauer
Associates, Sunderland, Mass., 2003).

three dimensional pump) [53]. It has been proposed that the heart evolved through
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modification and expansion of an ancestral network of regulatory genes encoding a core set
of cardiac transcription factors, including NK2, myocyte enhancer factor 2 (MEF2), GATA,
Tbx and Hand, virtually all of which are present in triploblasts [39, 61]. The ancestral circuit
was probably responsible for the contractile mesothelial cell that lined the earliest blood
vessels and ultimately acquired a contractile phenotype to propel the blood. Complexity of
the heart increased during evolution owing to the expansion of the number of ancestral
regulatory genes, modification of the timing and pattern of their expression, as well as their
regulatory interactions with each other and with other regulatory inputs.
What is the origin of the endothelial cell? One possibility is that it arose from the
mesothelial cell. Alternatively, endothelial cells are derived from amoebocytes, through the
acquisition of an epithelial phenotype [55, 62]. A previous study demonstrated that
amoebocytes in amphioxus larvae express Pdvegfr (a single member of the platelet-derived
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growth factor receptor [PDGFR]/ vascular endothelial growth factor receptor [VEGFR]
subfamily) [39]. The authors proposed that vertebrate endothelial cells may have derived
from ancestral free hemal Pdvegfr+ cells [39]. Consistent with an amoebocyte origin of
endothelial cells is the observation that endothelial cells arise in close association with
hematopoietic cells. For example, they share a common progenitor cell (the hemangioblast)
and some hematopoietic cells are derived from hemogenic endothelium. It also noteworthy
that hemocytes in Drosophila express VEGFR [63].
As in the case of the heart, the endothelium likely arose through the mutation and selection
of a small number of pre-existing regulatory genes involved in mesoderm function. It is, of
course, highly improbable that selection has coordinated hundreds of different DNA
mutations to yield such a broad array of endothelial cell phenotypes [57]. Instead, the
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endothelium has been selected for its plasticity and exploratory behavior [64]. As a result,
organs can acquire changes without depending on co-evolution in the endothelium.
The ancestral vertebrate developed the means to assemble endothelial cells into tubes, to
recruit pericytes and stabilize blood vessels, and to specify different subtypes of vessels,
including arteries and veins. The extent to which the processes “borrowed” from established
building plans is unclear. It is notable that VEGF-like factors have been identified in a
number of invertebrates, including drosophila [63], and squid [65]. Moreover, a blueprint for
tubulogenesis exists in multiple systems, including the tracheal system of insects [66, 67].
The formation of blood vessels in the closed circulatory system of invertebrates has been
referred to as “myoepithelial angiogenesis” [51]. The process may involve hemocyte-
mediated clearance of extracellular matrix to form a patent lumen [38, 62], or the separation
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of apposing mesothelial basement membranes, perhaps via cell-cell repulsion [62]. Studies
in Drosophila have implicated a role for Robo-Slit in heart tube formation [62].
EVOLUTIONARY MEDICINE
In the Introduction, we emphasized the importance of both proximate and evolutionary
explanations when approaching human physiology and pathophysiology. Evolutionary
medicine is the application of evolutionary principles to an understanding of human health

and disease [1, 68, 69]. Evolutionary biology teaches us that no trait is perfect. Every trait
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can be made better, but by making it better something else would be made worse. Indeed, a
central tenet of evolutionary medicine is that the human body is a jerry-rigged bundle of
trade-offs and that an understanding of the cost-benefits of these trade-offs will provide
novel insights into our vulnerability to disease. For example, consider the closed circulatory
system. Such a design permits more efficient bulk flow delivery to the tissues of the body.
However, a closed vasculature is by definition branched and curved. Blood flow is disturbed
at branch points and curvatures. Microdomains of flow disturbance render the endothelium
dysfunctional and vulnerable to atherosclerosis. This is a classic example of an evolutionary
tradeoff, where the advantages of closing the circulation are balanced against the
disadvantage of increased susceptibility to atherosclerotic lesion formation. Another
important evolutionary principle when considering human health and disease is the notion
that the present-day blood vascular system evolved to maximize fitness in a far earlier era,
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perhaps tens of thousands of years ago, which is the time it takes for natural selection to
filter the gene pool. Our evolved state has not had time to adapt to changes in longevity,
physical activity, diet and recreational drug use (especially smoking). The resulting gene-
environment mismatch has led to an epidemic of hypertension, hypercholesterolemia,
diabetes and atherosclerosis. A goal of preventive medicine is to recalibrate the balance by
adapting the environment to the genetic blueprint.
CONCLUSIONS
Clinicians and clinician-scientists tend to view the cardiovascular system through the narrow
lens of proximate causation. In this review, we have attempted to place the blood vascular
system in a broader temporal context, with the goal of emphasizing the selection pressures
that have led to its emergence. An important take home message is that cardiovascular
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systems are highly diverse in their structure and function. The design of a given system is
exquisitely matched to the needs of the animal. It is not helpful to think of one system being
“superior” to another. Mollusks have no more need for a human heart, than humans have for
a mollusk heart. All extant animals have survived the rigor of natural selection and are by
definition highly successful. An appreciation of the particular adaptations that underlie these
many successes provides important insights into basic principles of oxygen delivery and
homeostasis in all animals, including humans.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments
We thank Steve Moskowitz for his artwork, Ellen Morgan and Kiichiro Yano for their technical help, and Dena
Groutsis for her help in procuring specimens. This work was supported by National Institutes of Health grant
HL076540. Author contributions: RME designed and performed experiments and analyzed the data. AMD designed
and performed experiments and analyzed the data. WCA designed and performed experiments, analyzed the data
and wrote the manuscript.

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Fig. 1. Metazoan phylogenetic tree

Adapted and modified from Juliano CE, Swartz SZ, Wessel GM. A conserved germline
multipotency program. Development. 2010; 137:4113-26.
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Fig. 2.
Schematic of different circulatory systems.
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Fig. 3. Blood vascular system of the lobster

A, Schematic of the blood vascular system of the lobster. a, artery. B, Open view of the
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dorsal thorax showing the single-chambered heart suspended in the pericardial sinus by alary
ligaments. C, Electron micrograph of the heart showing a cardiomyocyte (cm) filled with
cross-sections of thick and thin microfibrils (consistent with myosin and actin) and
mitochondria (*). The inner surface of the heart chamber is covered by a thin basal lamina.
There is no endocardial lining. A hemocyte (h) is shown in the heart chamber. D, One-
micron transverse histological section of a dorsal abdominal artery stained with Giemsa. E,
Electron micrograph of the dorsal abdominal artery shows the wall consisting of
extracellular matrix composed of a dense meshwork of collagen fibrils. There is no
endothelial lining. F, Photomicrograph of a swimmeret of a lobster that has been injected
with Evans blue dye in the dorsal abdominal artery. Note the branching network of vessels
ending in plumes of extravasated dye in the interstitial space. Panel A is adapted
McLaughlin PA. Internal Anatomy. In LH Mantel (Ed.), The biology of crustacea: Internal
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anatomy and physiological regualtion. New York, NY: Academic Press, 1983.

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Fig. 4. Blood vascular system of the earthworm

A, Schematic of the blood vascular system of the earthworm. In this case, all vessels are
colored red since there is no specialized respiratory organ where blood is oxygenated and
because there is no central heart that divides vessels into afferent channels (veins) and
efferent channels (arteries). B, Transverse histological section through the body of the
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earthworm shows the dorsal vessel (arrow) on the dorsal side of the gut. The section was
stained with H&E. C, One-micron histological section stained with Giemsa shows two small
blood vessels in the body wall. Ep, epidermis; mu, circular muscle layer. D, Electron
micrograph of a small blood vessel surrounded by a continuous layer of myoepithelial cells.
The lumen is filled with hemoglobin particles. E, Higher power electron micrograph shows a
blood vessel lined by several myoepithelial cells. The cells are connected by specialized
lateral borders. They contain numerous thick myofilaments (arrows) consistent with myosin

that are oriented circumferentially around the vessel. A well-formed basal lamina (*)
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separates the myoepithelial cells from the lumen of the blood vessel. The fact that the
hemoglobin particles are retained in the lumen indicates that the basal lamina forms an
effective barrier. F, A similar electron micrograph to E, but shows the presence of an
amoebocyte in the blood vessel lumen adjacent to the myoepithlial lining. Panel A is
adapted from Brusca R.C., Brusca G.J. Invertebrates. Sunderland, MA: Sinauer Associates,
2003.
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Fig. 5. Blood vascular system of amphioxus

A, Schematic of the blood vascular system of amphioxus. B, Transverse histological section
through the pharynx shows the paired dorsal aortae (arrows) lying on either side of and
ventral to the notochord (NC). The hepatic vein is on the dorsal surface of the liver (also
referred to as the hepatic cecum). The section was stained with H&E. P, pharynx. C, One-
micron histological section stained with Giemsa shows a discontinuous coverage of dorsal
aorta with amoebocytes (arrows). D, Electron micrograph of the contractile hepatic vein
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shows the lumen delimited by parts of three myoepithelial cells, which contain
myofilaments (*) in their basal portion. a, amoebocyte. E, Electron micrograph of a skeletal
vessel in the gill surrounded by skeletal rod (skr). The skeletal rod, in turn is surrounded by
atrial epithelial cells (ae) and lateral ciliated cells (lcc). Occasional chromaffin-like cells
(with membrane-delimited electron dense granules) are scattered between the atrial
epithelial cells. Note the presence of amoebocytes (a) on the luminal surface of the blood
vessel wall. F, Electron micrograph shows an amoebocyte “clinging” to the luminal surface

of the dorsal aorta. The cell contains a well-developed Golgi apparatus (g), numerous
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vesicles, and granules containing tubular structures (arrows). Note that the amoebocyte has
no underling basal lamina. Rather it is applied to the connective tissue matrix. G, Higher
magnification of an amoebocyte from dorsal aorta shows two tubule-filled granules.
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Fig. 6. Blood vascular system of the hagfish

A, Schematic of the blood vascular system of hagfish. Top, Transverse section through mid-
region shows several features that are typical of other vertebrates, including the arrangement
of myomeres, neural tube, and aorta. Features that are unique to hagfish include the large
subcutaneous sinus between skin and skeletal muscle, the retention of the notochord in the
adult, and the presence of slime glands on the ventrolateral surface. Bottom, schematic of
hagfish circulation. The gills are in series with the systemic circulation. Blood enters the
subcutaneous sinus via skeletal muscle capillaries and renters the systemic circulation via
accessory hearts (the caudal and cardinal hearts). The portal heart pumps blood from the
intestinal vasculature into the systemic heart via the common portal vein. B,
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Photomicrograph of ventral aorta and two (of a total of 12) gills in an animal that has been
injected with Evans blue dye through the heart. C, Electron micrograph of the heart shows
electron-lucent endothelial cells (EC) overlying a thick extracellular matrix containing a
chromaffin-like cell, and a cardiomyocyte (cm) with electron-lucent cytoplasm, and well-
preserved mitochondria and muscle filaments. D, Electron micrograph of the dermis shows a
microvessel in cross-section containing two nucleated red blood cells. The blood vessel is
lined by a continuous layer of endothelial cells. A melanocyte is seen on the left side of the

vessel. E, Electron micrograph of a kidney glomerulus shows podocyte foot processes

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abutting a well formed basal lamina. On the other side of the basal lamina is an endothelial
cell (EC) facing the lumen of a glomerular capillary. The endothelial cell contains many
vesicles, vacuoles and tubular structures. F, Electron micrograph of a liver sinusoid shows a
large gap in a single endothelial cell (E1, double-headed arrow), well-preserved attachments
between two endothelial cells (EC1 and EC2), and a continuum of proteinaceous material
from the lumen to extravascular space (Space of Disse). EC2 is a second endothelial cell.
Panel A is reprinted with permission from Cheruvu PK, Gale D, Dvorak AM, Haig D, Aird
WC. Hagfish: a model for early endothelium. In W Aird (Ed.), Endothelial Biomedcine.
Cambridge, UK; New York: Cambridge University Press, 2007. Panels C, D and F are
reprinted with permission from Yano K, Gale D, Massberg S, Cheruvu PK, Monahan-Earley
R, Morgan ES, Haig D, von Andrian UH, Dvorak AM, Aird WC. Phenotypic heterogeneity
is an evolutionarily conserved feature of the endothelium. Blood. 2007;109:613-5.
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Fig. 7. Phylogenetic perspective of the blood vascular system

Blood vascular system types and major propulsive organs are shown for representative
extant phyla. The phylogenetic tree is schematic only, and the timelines are not drawn to
scale. EC, endothelial cell; mya, mullion years ago.
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Evolutionary origins of the blood vascular system and

3Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215

as a means to overcome the time-distance constraints of diffusion. The endothelium evolved in an

dysfunctional in disease? By contrast, little attention is paid to the evolutionary mechanisms

introduce primary ultrastructural data) from a spectrum of representative species, with a

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

(including cephalocordates, urochordates and vertebrates) as well as hemichordates (acorn

disproportionately changes the ratio of surface area to volume. Specifically, as a solid 3-

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

triploblasts, however, circulatory fluid is an internal, extracellular, aqueous medium

Coelomic circulatory systems—Some small and flat triploblastic animals (e.g.,

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

Blood vascular systems—The blood vascular system consists of blood-filled spaces

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

Closed circulatory systems occur in a wide variety of invertebrates including annelids,

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

invertebrates employ specialized invaginated structures, including book lungs (spiders) or

cardiovascular design in the animal kingdom. We begin with an example of an open

Case study 1: The lobster (Homarus americanus)

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

Case study 2: The earthworm (Lumbricus terrestris)

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In summary, the earthworm provides an example of an invertebrate with a closed blood

Case study 3: Amphioxus (Branchiostoma lanceolatum)

Amphioxus belongs to the phylum Chordata, subphylum Cephalochordata, which is the

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structure for the vessel wall [12].

Hagfish (Myxine glutinosa)

J Thromb Haemost. Author manuscript; available in PMC 2017 April 03.

In addition to vascular bed-specific differences in ultrastructure, the endothelium of hagfish

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this section, we will consider each question in turn.

endothelium is present in all vertebrates, yet distinctly absent in invertebrates. Although

Why did the blood vascular system and endothelium evolve?

mechanism. More importantly, they became increasingly localized to distinct compartments

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improved distribution of nutrients absorbed by the intestine [51]. Myoepithelial

between myocytes (hence, simultaneous contraction) and one-way valves [53].

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How did the blood vascular system and endothelium evolve?

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21. Reiber C, McMahon B, Burggren W. Cardiovascular functions in two macruran decapod

151:467–72. S1532-0456(10)00024-4 [pii]. DOI: 10.1016/j.cbpc.2010.02.003 [PubMed:

(Branchiostoma lanceolatum Pallas). J Exp Biol. 2000; 203:3381–90. [PubMed: 11044377]

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blood-2006-05-026401 [PubMed: 16990601]

Hemost. 2010; 36:276–85. DOI: 10.1055/s-0030-1253450 [PubMed: 20490978]

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Fig. 1. Metazoan phylogenetic tree

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Fig. 3. Blood vascular system of the lobster

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Fig. 4. Blood vascular system of the earthworm

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