Fenestrae Biogenesis

The inability to maintain fenestrated endothelium in tissue culture and the difficulty in quantifying and manipulating the appearance of fenestrae both in vivo and in vitro have greatly hampered the study of their biogenesis. Until recently, the induction of fenestrae in cultured endothelial cell lines has been reported to yield numbers that are three to four orders of magnitude lower in density than normally observed in vivo. Nevertheless, a small number of key studies published throughout the last three decades have highlighted several extracellular and intracellular determinants that may be involved in the differentiation program of a fen-estrated endothelial cell.

Endothelial Cell Microenvironment

Fenestrated microvessels are in constant contact with the extracellular matrix, both in the form of routine basal lamina or, in some cases, elaborate, thickened matrix such as the basement membrane of the kidney glomerulus or the multilamellate Bruch's membrane, which separates pigmented retina from the choriocapillaris (Figure 4). Extracellular matrix could simply provide a structural scaffold to facilitate the extreme shape changes and cell attenuation that accompanies the fenestrated phenotype. However, a more active, instructive role for the extracellular matrix has been proposed to explain, for example, the presence of fenestrae only in the regions of the choriocapillaris that are immediately adjacent to Bruch's membrane. In vitro experiments examining the effects of a variety of extracellular matrices suggest that fenestrae formation is supported by specific matrix components, which mimic the situation observed in vivo.

Signal Transduction Pathways

The first attempts to reprogram the fenestrae differentiation program in cultured endothelial cells relied on the use of potent and relatively nonspecific initiators of intracellular signaling cascades on isolated bovine adrenal cortex endothelial cells. Phorbol myristate acetate, an activator of protein kinase C isoforms and a potent differentiation agent for some cell types, promoted a change in the shape of endothelial cells accompanied by a five-fold increase in the frequency of diaphragmed fenestrae (approximately six fenestrae per 100 mm2). Similarly, treatment of cultures with retinoic acid led to a threefold increase in the surface density of fenestrae, while transforming growth factor b led to a sevenfold decrease in their density. The physiological relevance of these signaling pathways remains to be established.

The angiogenic growth factor, vascular endothelial growth factor (VEGF), is the strongest candidate for a signaling protein that induces fenestrae formation. VEGF is an endothelial-specific mitogen and motogen, and was originally identified as a vascular permeability factor, approximately 50,000 times more potent than histamine. Although VEGF is greatly downregulated after the completion of embryonic vasculogenesis and angiogenesis, it is continuously and highly expressed in epithelial cells adjacent to fenestrated endothelium. Moreover, fenestrae have been

Figure 2 Schematic representation of a capillary with fenestrated endothelium and underlying basement membrane. Fenestrae are found only at the most attenuated regions of endothelial cells, where the nucleus and organelles are excluded and the distance from apical to basal plasma membrane is as little as 40 nm. They occur in groups and are arranged in a near-linear fashion with precise spacing between them. Inset shows the fenestral pore to be 60 nm in diameter, with an effective size of 5 to 6nm when apertured by a diaphragm. [Adapted from Rhodin (1962), J. Ultrastructure Res. 6, 171-185.] (see color insert)

Figure 2 Schematic representation of a capillary with fenestrated endothelium and underlying basement membrane. Fenestrae are found only at the most attenuated regions of endothelial cells, where the nucleus and organelles are excluded and the distance from apical to basal plasma membrane is as little as 40 nm. They occur in groups and are arranged in a near-linear fashion with precise spacing between them. Inset shows the fenestral pore to be 60 nm in diameter, with an effective size of 5 to 6nm when apertured by a diaphragm. [Adapted from Rhodin (1962), J. Ultrastructure Res. 6, 171-185.] (see color insert)

observed in the neovasculature of tumors and in the normally continuous endothelium of the retina during diabetic microangiopathy, both pathological situations that are functionally linked to the local upregulation of VEGF. In fact, in vivo studies have demonstrated that VEGF can induce fenestrae within 10 minutes in the continuous endothelium of skeletal muscle and skin, when applied topically or injected intradermally, and in vitro studies using capillary endothe-lial cells further reinforce the ability of VEGF to promote fenestrae biogenesis. It is still unknown to what degree overall VEGF-induced permeability can be attributed to fenes-trae formation, since VEGF also triggers the appearance of other structures implicated in permeability, such as caveolae and vesiculo-vacuolar organelles (VVOs), and regulates the gating properties of endothelial junctions.

Cytoskeleton

The importance of cytoskeletal remodeling in fenestrae biogenesis has been suggested from in vitro studies examin-

Bruch Membrane Losses

Figure 3 Fenestral diaphragms revealed by quick-freezing and deep-etching of rat kidney peritubular capillaries. The intertwining fibrils bridging the pore and converging into a central mesh are apparent in this luminal view. Magnification: 120,000x. [Courtesy of Drs. Bearer and Orci (1985), J. Cell. Biol. 100, 418-428.] (see color insert)

Figure 3 Fenestral diaphragms revealed by quick-freezing and deep-etching of rat kidney peritubular capillaries. The intertwining fibrils bridging the pore and converging into a central mesh are apparent in this luminal view. Magnification: 120,000x. [Courtesy of Drs. Bearer and Orci (1985), J. Cell. Biol. 100, 418-428.] (see color insert)

Figure 4 Fenestrated endothelium of choriocapillaris. This image reveals the close association of diaphragmed pores to Bruch's membrane (BM) and microvillar base of retinal pigmented epithelium (RPE). (We thank associate Eunice Cheung for this data.) (see color insert)

ing the effect of agents that disrupt actin microfilament assembly on highly fenestrated liver sinusoidal endothelial cells. Latrunculin A, which depolymerizes actin filaments through the sequestration of actin monomers, as well as Cytochalasin B, which leads to disassembly by capping the fast-growing end of actin filaments, both led to a two- to threefold increase in the number of fenestrae within 30 to 60 minutes. The physiological relevance of this data was recently supported by experiments showing that a dominant negative version of the small GTP-binding protein Rac could block VEGF-driven fenestrae formation during corneal angiogenesis, putatively through interfering with the reorganization of the actin cytoskeleton.

General Concepts for Fenestrae Formation

Researchers have used the limited amount of experimental data to generate several conceptual models (not mutually exclusive) to guide research in the area of fenes-trae biogenesis.

Caveolae Give Rise to Fenestrae

Fenestrae and caveolae share structural features, common tissue distribution, a relationship to the VEGF signaling cascade, and a common putative role in the regulation of vascular permeability. in one of the earliest hypotheses for fenestrae formation, investigators postulated that caveolae may give rise to fenestrae in a process involving fusion of a budding caveolar vesicle with the adjacent plasmalemma. The discovery that PV-1 was common to the diaphragms that reside in both fenestrae and the caveolae of endothelial cells further supported this model. Current evidence, however, emphasizes more differences than similarities in the nature of the two organelles. General compositional differences between the two organelles were first highlighted in tracer perfusion studies showing that caveolar diaphragms

(also referred to as stomatal diaphragms) lacked anionic sites among other molecular determinants that were present within the fenestral diaphragm. Furthermore, exclusion of the main structural component of caveolae, caveolin-1, from fenestrae in vivo and in vitro, and the recent finding that knockout mice completely lacking caveolae still have fenes-trae, provide compelling evidence for distinct origins of caveolae and fenestrae. However, the existence of a common structural precursor that differentiates to give rise independently to fenestrae and caveolae still remains a valid point for consideration.

A Putative Role for the Diaphragm in Fenestrae Formation

The presence of a diaphragm in fenestrae is variable; it is found in the fenestrated capillaries of the intestine, the choriocapillaris, the choroid plexus, and endocrine organs, while it is absent from the more permeable microvessels of the kidney glomerulus and the liver sinusoids. Diaphragmed fenestrae, however, do initially appear within the vessels of developing fetal glomerulus, which has raised speculation on a role for the diaphragm in fenestrae biogenesis, in addition to its putative role in gating the pore. Achieving a precise circular opening and facilitating the extreme membrane curvature at the rim of fenestrae, where apical and basal plasma membranes come together, could be functions of the protein-rich diaphragm.

Apical-Basal Plasma Membrane Fusion Following Actin Displacement

Data demonstrating that actin microfilament disassembly triggers fenestrae biogenesis have spawned a model whereby removal of the actin-rich cortex beneath the plasma membrane is required to allow close apposition and fusion of apical and basal plasma membranes. Cortical actin removal has been shown to be an important prerequisite to membrane fusion during exocytosis, while removal of organelle-bound actin has been suggested to accelerate mammalian endosome and yeast vacuole membrane fusion. Whether the cytoskeleton also plays an instructive role in fenestrae formation remains to be established.

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

Get My Free Ebook


Post a comment