Vascular endothelium is a highly differentiated cellular monolayer with the organization
of a simple squamous epithelium. It lines the entire cardiovascular system and thus
constitutes a quasi-ubiquitous presence in organs and tissues throughout the body.
One of the most significant aspects of the vascular endothelium is its diversity of
phenotype and function, which endows it with the ability to regulate the exchange of
myriad substances between blood plasma and interstitial fluid in all tissues of the body.
The ability of the endothelium to regulate the transendothelial exchange or vascular
permeability is critically important in the growth, maintenance, and survival of all
tissues, as well as in the delivery of therapies to correct locations.
Within the circulation, exchanges occur across the endothelium at all levels of the vascular tree.
However, two functional segments are recognized with respect to the role of the vessel wall
in the transendothelial exchanges between blood and tissues served:
the conduit vessels and the exchange vessels.
The conduit vessels take blood from heart to tissues (large arteries down to the smaller
muscular arterioles upstream of the precapillary sphincter) or from tissues to heart
(small muscular veins collecting from venules and draining into large veins).
In these vessels (with large sectional diameter but low aggregated exchange surface),
the transendothelial exchanges are thought to have impact only on the vessel wall and
not on the organs/tissues crossed by these vessels. The exchange vessels consist of
capillaries and venules, and are thought to be involved in active exchanges between blood and tissues.
This segment, which has low sectional surface per vessel (i.e. <100 µm diameter for venules and
<10-20 µm for capillaries) yet the largest aggregated surface, is characterized by a very thin wall
consisting of endothelium (with an average thickness of less than 0.3 µm) overlying basement membrane
and occasional pericytes (situated mostly in venules). The term "microvascular permeability",
as used extensively in the literature, describes the transendothelial exchange of fluids and solutes.
It is generally recognized that endothelium mediates "basal" microvascular permeability,
which refers to the steady, continuous transverse flow of fluid and hydrophilic solutes (both small
and large molecules) across the normal healthy microvascular endothelium, and "inducible"
or "increased" microvascular permeability, which refers to the increase in permeability
to fluid and plasma proteins that occurs in inflamed tissues or tumors. Although the mechanisms
underlying basal permeability remain poorly understood, previous investigations have provided important
insights into the molecular basis of inducible permeability.
A critical step in the evolution of multicellular organisms (metazoans and higher) was the establishment
of different biological compartments to carry out specific functions. This entailed the development of
epithelial cellular barriers between different environments as well as the development of strategies to
selectively move molecules between compartments (i.e., for delivery of nutrients, elimination of wastes
as well as ensuring molecular communication between distant cells and tissues) while maintaining their
distinct composition.
The movement of material is generally accomplished via routes situated either across (i.e., transcellular)
or in between (i.e., paracellular) the cells forming the barrier. While the paracellular transport occurs
via the intercellular junctions, there are at least three transcellular routes described so far:
(a) water and small molecules are moved via membrane transporters that have a different distribution/orientation
on opposite fronts of a barrier cell;
(b) transcytosis, defined as the transport of macromolecular cargo from one front of a polarized cell to the other
within membrane-bounded carrier(s), and (c) pores or channels which are patent openings through the barrier cells
with or without selective permeability for different molecules.
Together, these processes contribute to the success of multicellular organisms.
A) Pathways of exchange across epithelia. B) Schematic of transcytosis vs the pore theory of exchange across endothelia.
The transcellular exchange systems have achieved a high level of complexity and sophistication in mammals.
Among different types of epithelia, the vascular endothelium has the most diversified repertoire of specific
structures that might carry out these exchanges. During decades of research on the topic, several endothelial
subcellular structures that might participate in this function have been identified, such as endocytic vesicles
(i.e. caveolae), transendothelial channels (TEC), vesiculo-vacuolar organelles (VVO), fenestrae and intercellular junctions.
Questions we would like to answer:
a)
The identity and importance of vesicular pathways in the transport of macromolecules across endothelial barriers.
b)
What are the components, biogenesis, precise function and regulation of fenestrae, TEC and VVO as well as the
stomatal diaphragms of caveolae. Research on these structures was bolstered by our finding of PV-1 as the first
and only known marker of the stomatal diaphragms of caveolae, TEC and VVO and of fenestral diaphragms. First we
obtained evidence (including genetic evidence) that this molecule is indeed a key component of the endothelial diaphragms.
By using this gene product as a molecular handle we are unraveling the components of these structures and arrive at
insights on their biogenesis and regulation. Furthermore, some of our projects deal with devising new assays for evaluation
of their function.
Model of PV1 integration in the diaphragms of different structures involved in permeability
The increase in permeability in inflammation is thought to occurs in a defined segment of the circulation of a given
capillary bed, namely in the postcapillary venules. There are several theories in the field that postulate either
formation of gaps in between the cells or through the cells via VVO. As PV-1 is a component of VVO, we are interested
in determining whether these structures are involved in inflammation and under which conditions. Furthermore, PV-1 being
highly expressed on activated endothelium in inflammation and angiogenesis we are actively pursuing its function(s)
in such settings.
1)
Caveolae with (top right and left panel) and without (right bottom panel) stomatal diaphragms
2)
Vesiculo-vesicular organelle (VVO)
3)
Fenestrae with and without (glomerulus) fenestral diaphragms
4)
Transendothelial channels
5)
SEM of fenestrae
6)
Endothelial cells treated with latrunculin A. Staining with PV1(red), Cav1 (green) and actin (blue).
7)
In PV1 containing endothelial cells, cytoskeleton disruption by either latrunculin A or cytochalasin D leads to the formation of fenestrae disposed in sieve plates. Left - Staining with Phalloidin-Alexa 488 (green) and anti-PV1-Alexa 568 mAb (red) of cells treated (left panels) or untreated (right panels) with 2.5µM latrunculin A. Right - Staining with anti-tubulin Alexa 488 mAb (green) and anti-PV1-Alexa 568 mAb (red) of cells treated (left panels) or untreated (right panels) with 2.5µM latrunculin A. Fenestrae sieve plates are indicated with a white contour.
8)
Fenestrae formation induced in endothelial cells by PMA. Actin (red, Phalloidin-Alexa647) form bundles which causes the sieve plates (green - staining with anti-PV1-Alexa488 mAb) to be smaller.
1)
Bird eye view of cells labeled with PV1 and caveolin 1
2)
PV1 moves with Caveolin 1