A thick endothelial surface coat consisting of the glycocalyx and associated plasma proteins has been hypothesized to reduce functional capillary volume available for flowing plasma macromolecules ...and blood cells. The purpose of this study was to compare anatomic and functional capillary diameters available for macromolecules, RBCs, and WBCs in hamster cremaster muscle capillaries. Bright-field and fluorescence microscopy provided similar estimates (mean +/- SE) of the anatomic capillary diameter: 5.1 +/- 0.1 microns (bright field, 39 capillaries in 10 animals) and 5.1 +/- 0.2 microns (membrane dye PKH26, 18 capillaries in 2 animals). Estimates of functional diameters were obtained by measuring the width of RBCs and WBCs and the intracapillary distribution of systemically injected fluorescein isothiocyanate (FITC)-dextran 70. WBCs (5.1 +/- 0.2 microns) fully occupied the anatomic capillary cross section. In contrast, the widths of RBCs (3.9 +/- 0.2 microns, 21 capillaries in 8 animals) and FITC-dextran (4.3 +/- 0.2 microns, 21 capillaries in 8 animals) were significantly smaller than the anatomic capillary diameter. Continuous (1- to 5-minute) excitation of fluorochromes in the capillary lumen (light-dye treatment) increased the width of RBCs passing the treated site from 3.6 +/- 0.3 to 4.4 +/- 0.3 microns (6 capillaries in 4 animals) and the width of the FITC-dextran column from 4.1 +/- 0.2 to 4.6 +/- 0.3 microns (10 capillaries in 7 animals). Furthermore, light-dye treatment increased capillary tube hematocrit by 60% in 40-microns-long capillary segments compared with untreated sites in the same capillaries. It is concluded that the wall of skeletal muscle capillaries is decorated with a 0.4- to 0.5-microns-thick endothelial surface coat, which may represent the true active interface between blood and the capillary wall.
Connexin 43 (Cx43) is a protein expressed in a variety of mammalian tissues. However, the lack of specific blockers and the absence of known genetic mutants have hampered the investigation of the ...function of this protein. Cx43-null mice die shortly after birth, thus preventing functional studies in vivo. Here, we report the generation and characterization of a vascular endothelial cell-specific deletion of the Cx43 gene (VEC Cx43 KO) in mice by using the loxP/Cre system. Using homologous recombination, a mouse line was created carrying loxP sites flanking exon 2 of the Cx43 gene ("floxed" mice). To produce cell specific deletion of the Cx43 gene, these mice were crossed with animals from a line carrying the Tie 2-Cre transgene. The homozygous VEC Cx43 KO mice survived to maturity. However, they were hypotensive and bradycardic when compared with heterozygous VEC Cx43 KO mice, or to the floxed Cx43 gene mice. The hypotension was associated with marked elevation of plasma nitric oxide (NO) levels as well as elevated plasma angiotensin (Ang) I and II. We hypothesize that endothelial cell Cx43 plays a key role in the formation and/or action of NO, and that the elevation of Ang II is a secondary event. The specific cellular basis for the hypotension remains to be established, but our findings support the idea that endothelial Cx43 gap junctions are involved in maintaining normal vascular function; moreover, these animals provide the opportunity to determine more clearly the role of endothelial Cx43 in vascular development and homeostasis.
It is well known that vascular smooth muscle tone can be modulated by signals arising in the endothelium (e.g., endothelium-derived relaxing factor, endothelium-derived hyperpolarizing factor, and ...prostaglandins). Here we show that during vasoconstriction a signal can originate in smooth muscle cells and act on the endothelium to cause synthesis of endothelium-derived relaxing factor. We studied responses to two vasoconstrictors (phenylephrine and KCl) that act by initiating a rise in smooth muscle cell intracellular Ca2+ concentration Ca2+i and exert little or no direct effect on the endothelium. Fluo-3 was used as a Ca2+ indicator in either smooth muscle or endothelial cells of arterioles from the hamster cheek pouch. Phenylephrine and KCl caused the expected rise in smooth muscle cell Ca2+i that was accompanied by an elevation in endothelial cell Ca2+i. The rise in endothelial cell Ca2+i was followed by increased synthesis of NO, as evidenced by an enhancement of the vasoconstriction induced by both agents after blockade of NO synthesis. The molecule involved in signal transmission from smooth muscle to endothelium is as yet unknown. However, given that myoendothelial cell junctions are frequent in these vessels, we hypothesize that the rise in smooth muscle cell Ca2+ generates a diffusion gradient that drives Ca2+ through myoendothelial cell junctions and into the endothelial cells, thereby initiating the synthesis of NO.
We previously reported that a 0.4- to 0.5-microm-thick endothelial surface layer confines Dextran 70 (70 kDa) to the central core of hamster cremaster muscle capillaries. In the present study we used ...a variety of plasma tracers to probe the barrier properties of the endothelial surface layer using combined fluorescence and brightfield intravital microscopy. No permeation of the endothelial surface layer was observed for either neutral or anionic dextrans >/=70 kDa, but a neutral Dextran 40 (40 kDa) and neutral free dye (rhodamine, 0.4 kDa) equilibrated with the endothelial surface layer within 1 min. In contrast, small anionic tracers of similar size (0. 4-40 kDa) permeated the endothelial surface layer relatively slowly with half-times (tau(50)) between 11 and 60 min, depending on tracer size. Furthermore, two plasma proteins, fibrinogen (340 kDa) and albumin (67 kDa), moved slowly into the endothelial surface layer at the same rates, despite greatly differing sizes (tau(50) approximately 40 min). Dextran 70, which did not enter the glycocalyx over the course of these experiments, entered at the same rate as free albumin when it was conjugated to albumin. These findings demonstrate that for anionic molecules size and charge have a profound effect on the penetration rate into the glycocalyx. The equal rates of penetration of the glycocalyx demonstrated by the different protein molecules suggests that multiple factors may influence the penetration of the barrier, including molecular size, charge, and structure.
The endothelial cell glycocalyx influences blood flow and presents a selective barrier to movement of macromolecules from plasma to the endothelial surface. In the hamster cremaster microcirculation, ...FITC-labeled Dextran 70 and larger molecules are excluded from a region extending almost 0.5 micrometer from the endothelial surface into the lumen. Red blood cells under normal flow conditions are excluded from a region extending even farther into the lumen. Examination of cultured endothelial cells has shown that the glycocalyx contains hyaluronan, a glycosaminoglycan which is known to create matrices with molecular sieving properties. To test the hypothesis that hyaluronan might be involved in establishing the permeation properties of the apical surface glycocalyx in vivo, hamster microvessels in the cremaster muscle were visualized using video microscopy. After infusion of one of several FITC-dextrans (70, 145, 580, and 2,000 kDa) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent dextran columns and of red blood cell columns (means +/- SE). The widths of the red blood cell and dextran exclusion zones were calculated as one-half the difference between the bright-field anatomic diameter and the width of the red blood cell column or dextran column. After 1 h of treatment with active Streptomyces hyaluronidase, there was a significant increase in access of 70- and 145-kDa FITC-dextrans to the space bounded by the apical glycocalyx, but no increase in access of the red blood cells or in the anatomic diameter in capillaries, arterioles, and venules. Hyaluronidase had no effect on access of FITC-Dextrans 580 and 2,000. Infusion of a mixture of hyaluronan and chondroitin sulfate after enzyme treatment reconstituted the glycocalyx, although treatment with either molecule separately had no effect. These results suggest that cell surface hyaluronan plays a role in regulating or establishing permeation of the apical glycocalyx to macromolecules. This finding and our prior observations suggest that hyaluronan and other glycoconjugates are required for assembly of the matrix on the endothelial surface. We hypothesize that hyaluronidase creates a more open matrix, enabling smaller dextran molecules to penetrate deeper into the glycocalyx.
Dye tracers were chosen, based on net charge, chemical structure, and reactive groups, to test for the existence of and to provide novel insight into channel selectivities of junctional pathways ...connecting smooth muscle and endothelial cells of the arteriolar wall. Dyes were injected into individual smooth muscle or endothelial cells of hamster cheek pouch arterioles using microiontophoresis. Coupling, independent of tracer net charge, was seen both within and between cell layers. Endothelial cells were well coupled by all of the tested dyes. Smooth muscle junctions appeared less effective in dye transfer than endothelial junctions. Lucifer yellow was confirmed to be a poor tracer of smooth muscle gap junctions, and remarkably this dye and other related sulfate-containing molecules interfered with dye movement through smooth muscle but not endothelial junctions. Myoendothelial junctions showed a striking polarity of dye movement, with dye transfer from endothelial to smooth muscle cells but little or no transfer in the reverse direction. Because the dyes have size and charge characteristics similar to those of known cellular second messengers, these findings have important implications for cell-cell signaling in the vessel wall.
The endothelial luminal glycocalyx has been largely ignored as a target in vascular pathophysiology even though it occupies a key location. As a model of the inflammatory response, we tested the ...hypothesis that tumor necrosis factor-alpha (TNF-alpha) can alter the properties of the endothelial apical glycocalyx. In the intact hamster cremaster microcirculation, fluorescein isothiocyanate (FITC)-labeled Dextrans 70, 580, and 2,000 kDa are excluded from a region extending from the endothelial surface almost 0.5 micrometer into the lumen. This exclusion zone defines the boundaries of the glycocalyx. Red blood cells (RBC) under normal flow conditions are excluded from a region extending even farther into the lumen. The cremaster microcirculation was pretreated with topical or intrascrotal applications of TNF-alpha. After infusion of FITC-dextran, FITC-albumin, or FITC-immunoglubulin G (IgG) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent tracer columns and of the RBC columns (means +/- SE). After 2 h of intrascrotal TNF-alpha exposure, there was a significant increase in access of FITC-Dextrans 70 and 580 to the space bounded by the apical glycocalyx in arterioles, capillaries, and venules, but no significant change in access of FITC-Dextran 2,000. The effects of TNF-alpha could be observed as early as 20 min after the onset of topical application. TNF-alpha treatment also significantly increased the penetration rate of FITC-Dextran 40, FITC-albumin, and FITC-IgG into the glycocalyx and caused a significant increase in the intraluminal volume occupied by flowing RBC. White blood cell adhesion increased during TNF-alpha application, and we used the selectin antagonist fucoidan to attenuate leukocyte adhesion during TNF-alpha stimulation. This did not inhibit the TNF-alpha-mediated increase in permeation of the glycocalyx. These results show that proinflammatory cytokines can cause disruption of the endothelial apical glycocalyx, leading to an increased macromolecular permeation in the absence of an increase in leukocyte recruitment.
The distributions of connexin 43 (Cx43) and connexin 40 (Cx40) in smooth muscle and endothelium of resistance vessels were examined using indirect immunofluorescence techniques coupled with confocal ...microscopy. Cx43 and Cx40 were found in smooth muscle and endothelium. Similar staining patterns were found in microvessel samples from brain and cremaster of the rat and from arterioles of the hamster cheek pouch. Double-labeling studies showed a high degree of colocalization of Cx40 with Cx43, suggesting the presence of multiple connexins within a single junctional plaque. Quantitative comparisons were made of the fluorescent patterns in the endothelium and smooth muscle of rat brain arterioles. Cx43 and Cx40 plaque diameters were 0.9 +/- 0.1 and 0.8 +/- 0.1 (SE) microns, respectively, in the endothelial layer and 0.5 +/- 0.1 and 0.5 +/- 0.1 microns, respectively, in the smooth muscle. There was no difference between mean plaque diameters of Cx43 and Cx40 in endothelium or smooth muscle. However, plaques were significantly larger in endothelium than in smooth muscle (P < 0.05). These findings demonstrate the potential for cell-cell communication in both cell types of the wall of arterioles from three different tissues. The data also suggest a greater level of coupling within the endothelium.
Conducted vasomotor responses are viewed as one mechanism that functionally integrates the microvasculature. It is hypothesized that the conducted vasomotor response is the result of an electrical ...current and its passive electrotonic spread along the length of a microvessel. We tested this hypothesis in isolated, unpressurized arterioles from the hamster cheek pouch using conventional intracellular membrane potential recording techniques. The mean resting membrane potential (RMP) was -67 mV. KCl and phenylephrine (PE) pulse-stimulation applied through micropipettes could both induce transient depolarizations and vasoconstrictions at the site of stimulation (local) and at conducted (560 microns) sites. It was noted, however, that the conducted vasomotor response could not be induced until the conducted electrical response exceeded a threshold of -45 mV for a minimum amount of time. The relationship between the amplitude of constriction and the amplitude-time area of depolarization above -45 mV was the same for local and conducted KCl and for conducted PE but was significantly different from that for local PE. Nifedipine greatly reduced the local and conducted mechanical but not electrical responses. Our results indicate that the conducted vasomotor responses are the result of the generation and subsequent conduction of electrical signals along the vessel but that the corresponding mechanical response occurs only when the electrical response exceeds a threshold level.
We have previously shown that conducted vasomotor responses follow patterns that are consistent with a passive spread of electrical current along the length of the arterioles (Xia and Duling, Am. J. ...Physiol. 269 (Heart Circ. Physiol. 38): H2022-H2030, 1995. In this study, we define the cells through which the current flows. Isolated arterioles of hamster cheek pouch were used. The mean resting membrane potential (RMP) for randomly sampled arteriolar cells was -67 mV. When cell types were identified by dye injection, the RMPs were -68 and -67 mV for smooth muscle (SM) and endothelium (EC), respectively. Pulses of KCl induced transient, monophasic depolarizations at the site of stimulation (local), which were conducted decrementally along the length of the arteriole over several millimeters. During electrical conduction, three patterns of responses could be observed, but identical patterns of the conducted electrical responses were always observed in SM and EC. Phenylephrine stimulation also caused transient local and conducted depolarizations in both SM and EC. As with KCl stimuli, shapes of conducted electrical responses were identical in records made in both cell types. The results suggest that SM and EC are electrically coupled both homocellularly and heterocellularly.
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