Brian R. Duling

Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries

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.

Longitudinal Gradients in Periarteriolar Oxygen Tension A Possible Mechanism For the Participation of Oxygen in Local Regulation of Blood Flow

The oxygen tension (Po2) on the external surface of arterioles between 8 and 100μ in diameter was measured with oxygen microcathodes (2 to 6μ diameter) in the suffused cheek pouch of hamsters and in the cremaster muscle of hamsters and rats anesthetized with pentobarbital and urethane. Intravascular Po2 was measured in 10 vessels and compared with extravascular Po2. Good agreement was found, with a mean difference of 1.4 ± 0.8 (SE) mm Hg. Significant longitudinal gradients were observed in periarteriolar Po2. Oxygen tension fell from 35 ± 4 mm Hg on the small arteries (ca. 80μ diameter) to 20 ± 3 mm Hg at the end of the terminal arterioles. These measurements were obtained with a suffusion solution Po2 of 39 ± 8 mm Hg, a tissue Po2 of 8 ± 2 mm Hg and femoral arterial blood Po2 of 69 mm Hg. When the suffusion solution Po2 was raised to 79 mm Hg, the resultant measurements were 42 ± 3 on the small arteries and 21 ± 3 mm Hg at the end of the terminal arterioles. Similar experiments were carried out while animals were breathing 95% oxygen and the Po2 of the cheek pouch suffusion solution was 39 mm Hg. Under these conditions, small artery Po2 was 152 ± 13 mm Hg and terminal arteriolar Po2 was 37 ± 9 mm Hg. Femoral artery blood Po2 was 427 ± 12 mm Hg. These data are consistent with the hypothesis that oxygen diffuses from the precapillary vessels and that intravascular Po2 falls progressively along the resistance vessels. This finding suggests a possible mechanism for the involvement of O2 in local regulation of blood flow.

Permeation of the luminal capillary glycocalyx is determined by hyaluronan

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.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 μm 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.

The behavior of sonicated albumin microbubbles within the microcirculation: A basis for their use during myocardial contrast echocardiography

The purpose of this study was to determine whether the behavior of sonicated albumin microbubbles accurately mimics red blood cell flow in the microcirculation and is thus consistent with their use as in vivo tracers of red blood cell flow during myocardial contrast echocardiography. Accordingly, microbubbles prepared from fluorescein-conjugated albumin and fluorescently labeled red blood cells were injected intravascularly in eight golden hamsters. Their intravascular distribution, velocities, arteriolar-to-venular transit and flux ratios at branch points were determined in the microcirculation of the cheek pouch. Albumin microbubbles (mean diameter, 4.9 +/- 3.6 microns) and red blood cells displayed a similar frequency of distribution across the arteriolar lumen (33% in the central 20% of the arterioles), and their arteriolar velocities were also similar (2.5 +/- 0.7 mm/sec and 2.3 +/- 0.7 mm/sec,p = NS). The mean velocities of microbubbles correlated well with those of red blood cells at baseline and after adenosine application (r = 0.97 and r = 0.89, respectively), as did the calculated maximum velocity (r = 0.98 and r = 0.80, baseline and adenosine, respectively). The velocity profiles across the lumen of the vessels for albumin microbubbles and red blood cells were similar at baseline and after adenosine-induced velocity changes. The flux ratios at branch points also correlated well (r = 0.92, p less than 0.001). Arteriolar-to-venular transit times of albumin microbubbles were similar to those of red blood cells in vessels ranging in size from 22 microns to 45 microns. We conclude that the behavior of albumin microbubbles in the microcirculation mimics that of red blood cells and supports their use as intravascular tracers of red blood cell flow during myocardial contrast echocardiography.

Microvascular hematocrit and red cell flow in resting and contracting striated muscle

Microvascular hematocrit and its possible relation to oxygen supply were systematically examined. We studied the red cell volume fraction (hematocrit) in arterial blood and in capillaries under a variety of circumstances. Control capillary hematocrit averaged 10.4 +/- 2.0% (SE) and arteriolar (14.2 micrometer ID) hematocrit averaged 13.9 +/- 1.2% in cremaster muscles of pentobarbital-anesthetized hamsters. Carotid artery hematocrit was 53.2 +/- 0.6%. The low microvessel hematocrit could not be entirely explained by a high red cell flux through arteriovenous channels other than capillaries (shunting). Hematocrit was not only low at rest, but varied with physiological stimuli. A 1-Hz muscle contraction increased capillary hematocrit to 18.5 +/- 2.4%, and maximal vasodilation induced a rise to 39.3 +/- 9.5%. The quantitative relations between capillary red cell flux, arterial hematocrit, and total blood flow could be explained by a two-element model of microvascular blood flow that incorporated a relatively slow-moving plasma layer (1.2 micrometer). Such a model would generate a low microvessel hematocrit and might reduce the diffusion capacity of individual capillaries, but would not reduce time-averaged red cell flux or alter steady-state vascular oxygen supply.

Dye Tracers Define Differential Endothelial and Smooth Muscle Coupling Patterns Within the Arteriolar Wall

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 preparation and use of the hamster cheek pouch for studies of the microcirculation

Abstract The cheek pouch is a useful microcirculatory preparation which has both advantages and disadvantages as outlined below and should be chosen for use with these characteristics in mind. There are several advantages to the use of the cheek pouch over other tissues. (1) The ease of access and relatively non-traumatic double layered preparation make it useful in low magnification studies requiring repeated observation of the same site. (2) The pouch is highly vascular and all classes of microcirculatory vessels can usually be seen within the field of the microscope. Thus, comparative studies on the various microvascular segments are possible. (3) The clarity and optical properties of the pouch are good when compared with other densely vascularized tissues. (4) The pouch possesses both skeletal muscle and cutaneous microcirculatory beds. Therefore, it is particularly useful for comparative studies. Also this arrangement allows one to study the effects of skeletal muscle stimulation on a vascular bed and to use the noncontractile portion of the pouch as a control site. The hamster cheek pouch also suffers from several disadvantages. (1) The animals must be anesthetized. (2) Fairly extensive dissection and clearing of the pouch is required for high resolution studies. (3) The pouch preparation is virtually alymphatic and therefore is not a suitable tissue for studies of water flux. (4) The nerve and blood supplies are diffuse and therefore the pouch is not well suited for perfusion studies or for denervation experiments.

Gap junctions in the control of vascular function.

Direct intercellular communication via gap junctions is critical in the control and coordination of vascular function. In the cardiovascular system, gap junctions are made up of one or more of four connexin proteins: Cx37, Cx40, Cx43, and Cx45. The expression of more than one gap-junction protein in the vasculature is not redundant. Rather, vascular connexins work in concert, first during the development of the cardiovascular system, and then in integrating smooth muscle and endothelial cell function, and in coordinating cell function along the length of the vessel wall. In addition, connexin-based channels have emerged as an important signaling pathway in the astrocyte-mediated neurovascular coupling. Direct electrical communication between endothelial cells and vascular smooth muscle cells via gap junctions is thought to play a relevant role in the control of vasomotor tone, providing the signaling pathway known as endothelium-derived hyperpolarizing factor (EDHF). Consistent with the importance of gap junctions in the regulation of vasomotor tone and arterial blood pressure, the expression of connexins is altered in diseases associated with vascular complications. In this review, we discuss the participation of connexin-based channels in the control of vascular function in physiologic and pathologic conditions, with a special emphasis on hypertension and diabetes.

Central Role of Connexin40 in the Propagation of Electrically Activated Vasodilation in Mouse Cremasteric Arterioles In Vivo

&NA; When a short segment of arteriole is stimulated, vasomotor responses spread bidirectionally along the vessel axis purportedly via gap junctions. We used connexin40 knockout (Cx40‐/‐) mice to study vasomotor responses induced by 10‐second trains of electrical stimulation (30 Hz, 1 ms, 30 to 50 V) in 2nd or 3rd order arterioles of the cremaster muscle. Measurements were made at the stimulation site (local) and at conducted sites (500, 1000, and 2000 &mgr;m upstream). In wild‐type (Cx40+/+) animals, electrical stimulation evoked a local vasoconstriction and a conducted vasodilation that spread very rapidly along the vessel length without detectable decay. In Cx40‐/‐ mice, the conducted dilation was converted into either vasoconstriction or a slowly developing vasodilation that decayed along the vessel length. Tetrodotoxin (TTX, 1 &mgr;mol/L) had no effect on the local vasoconstriction in either Cx40+/+ or Cx40‐/‐ mice, but enhanced the conducted vasodilation in Cx40+/+ animals. In Cx40‐/‐ mice, TTX abolished the conducted vasoconstriction when present and revealed a small vasodilation that decayed with distance. In the group of Cx40‐/‐ mice in which electrical stimulation elicited a conducted vasodilation, TTX had no effect. Immunocytochemistry revealed Cx40 only in the endothelial layer of arterioles from Cx40+/+ mice and complete elimination of this connexin in the Cx40‐/‐ animals. These results indicate that focal current stimulation causes vasoconstriction by a combination of perivascular nerve stimulation and smooth muscle activation. Moreover, electrical stimulation activates a nonneuronal, Cx40‐dependent vasodilator response that spreads along the vessel length without decay. (Circ Res. 2003;92:793–800.)

Microvascular Responses to Alterations in Oxygen Tension

The changes in microvascular diameter and perivascular oxygen tension resulting from alterations in suffusion solution Po2 were investigated in a study of the participation of oxygen in the regulation of blood flow. Diffusion gradients for oxygen were altered by changing the Po2 of a solution covering the surface of the hamster cheek pouch. As the solution Po2 was raised from a low of 11 mm Hg, perivascular Po2 of the large arterioles initially decreased to a minimum at approximately 40 mm Hg and then increased progressively as solution Po2 was elevated further. Arterial capillary and tissue Po2 remained relatively constant over a range of solution oxygen tensions between 11 and 40 mm Hg, suggesting that either the precapillary sphincters or the terminal arterioles were active in regulating tissue Po2 as the input of O2 from the solution was increased. The arterioles constricted as solution Po2 was elevated. Average arteriolar diameter decreased by 13% as solution Po2 increased from 11 to 47 mm Hg. A more pronounced constriction of 20% occurred when solution Po2 was increased from 11 to 84 mm Hg. These experiments indicated that the response of large and small arterioles was not mediated by a direct effect of oxygen on the vascular smooth muscle, since decreases in perivascular oxygen tension were coincident with decreases in vascular diameter in these vessels over a range of solution Po2 between 11 and 47 mm Hg. The data did not distinguish between a direct and an indirect effect of oxygen on the vascular smooth muscle of the terminal arterioles and precapillary sphincters. However, the oxygen tensions measured at these sites (18−30 mm Hg) required that the vascular smooth muscle cells respond to altered oxygen tension at levels higher than those which have been demonstrated experimentally.