Gerald A. Meininger

Regulation of tissue injury responses by the exposure of matricryptic sites within extracellular matrix molecules.

Extracellular matrix (ECM) is known to provide signals controlling cell shape, migration, proliferation, differentiation, morphogenesis, and survival. Recent data shows that some of these signals are derived from biologically active cryptic sites within matrix molecules (matricryptic sites) that are revealed after structural or conformational alteration of these molecules. We propose the name, matricryptins, for enzymatic fragments of ECM containing exposed matricryptic sites. Mechanisms regulating the exposure of matricryptic sites within ECM molecules include the major mechanism of enzymatic breakdown as well as others including ECM protein multimerization, adsorption to other molecules, cell-mediated mechanical forces, and ECM denaturation. Such matrix alterations occur during or as a result of tissue injury, and thus, the appearance of matricryptic sites within an injury site may provide important new signals to regulate the repair process. Here, we review the data supporting this concept and provide insight into why the increased exposure of matricryptic sites may be an important regulatory step in tissue responses to injury.

Vascular Smooth Muscle αvβ3 Integrin Mediates Arteriolar Vasodilation in Response to RGD Peptides

Arteriolar vasodilation and the resultant increase in blood flow are characteristic vascular responses to tissue injury. The dilatory mediators signaling these responses are incompletely understood. We show that integrin-binding peptides containing the Arg-Gly-Asp (RGD) tripeptide sequence cause immediate and, in some instances, sustained vasodilation when applied to isolated rat cremaster arterioles. The vasodilation is dependent on interaction of the soluble RGD sequence with the α v β 3 integrin expressed by smooth muscle cells in the arteriolar wall. Possible in vivo sources of soluble RGD sequences are fragments of extracellular matrix proteins that are generated after tissue injury. Indeed, protease-generated fragments of denatured collagen type I (a major source of RGD sequences) also cause cremaster arteriolar vasodilation through the α v β 3 integrin. Thus, extracellular matrix protein fragments containing the RGD sequence may act as vascular wound recognition signals to regulate blood flow to injured tissue.

Anatomic and hemodynamic characteristics of the blood vessels feeding the cremaster skeletal muscle in the rat

The anatomic arrangement, pressure distribution, and resting vascular tone of the feed arteries located upstream from the rat cremaster microcirculation were determined to characterize the sites of the vascular resistance in this macrovessel segment of the cremaster circulation. The cremaster microcirculation and its feeding arteries were studied using an intravital video microscopy system. Vascular diameters and pressures were measured with an image shearing monitor and servo-null micropipet system, respectively. The central arteriole of the cremaster muscle was found to be a distal segment of the external spermatic artery which branched from the pudic-epigastric artery that in turn arose from the common iliac artery. Together the length of these vessels, from the aorta to the cremaster muscle, was 37 mm and they accounted for 42% of the total pressure drop across the cremaster vascular network. The largest pressure drop (31 mm Hg) upstream from the cremaster occurred across the external spermatic artery which was also the longest (17.7 mm) feed vessel. Topical application of adenosine (1 X 10(-3) M) significantly dilated the pudic-epigastric artery and the external spermatic artery, indicating that these vessels had significant tone. In summary, our data indicate that the large fraction of network vascular resistance located in the feed vessels upstream from the cremaster is the result of both architectural features and vascular tone.

Myogenic vasoregulation overrides local metabolic control in resting rat skeletal muscle.

Microvascular reactions to increases in intravascular pressure were studied in the cremaster muscle of the anesthetized rat by enclosing the animal in an airtight box with the muscle exteriorized for observation of the microcirculation. Since the cremaster was exposed to atmospheric pressure, increasing pressure within the box produced equal increases in arterial and venous pressures. Thus, intravascular pressure was altered without affecting the pressure gradient for blood flow. Raising box pressure had no effect on respiration or heart rate and did not change the systemic activity of the sympathetic system, angiotensin II, or vasopressin. Diameters and flows were measured for first (107 +/- 3 micron, mean +/- SEM), second (87 +/- 5), third (29 +/- 2), and fourth (15 +/- 2) order arterioles during increases in intravascular pressure of +10, +20, and +30 mm Hg. No significant changes in the diameters of first or second order arterioles were elicited when pressure was increased. However, when box pressure was increased to +10, +20, or +30 mm Hg, a sustained constriction occurred in third (29%, 45%, and 63%, respectively) and fourth (5%, 38%, and 57%, respectively) order arterioles. Blood flow was significantly reduced in all arterioles, and perivascular PO2 was decreased adjacent to third and fourth order arterioles. Furthermore, the third order arteriole constrictor response was not abolished by local alpha-receptor blockade (phentolamine), indicating that it was not mediated by a local sympathetic axon reflex. Collectively, these data indicate that a potent, non-neural, pressure-dependent mechanism for vasoregulation is present in small arterioles of the cremaster. The sustained constriction in the presence of reduced blood flow and reduced periarteriolar oxygen tension indicates that the vascular response is independent of and capable of overriding flow-dependent (i.e., metabolic) control in resting skeletal muscle. The observations are compatible with the operation of a powerful myogenic mechanism in small arterioles.

Alteration of microtubule polymerization modulates arteriolar vasomotor tone.

Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 microM), nocodazole (10 microM), or demecolcine (10 microM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30-170 cmH(2)O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca(2+)](i).Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 μM), nocodazole (10 μM), or demecolcine (10 μM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30-170 cmH2O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca2+ concentration ([Ca2+]i). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca2+]i.

Altered cremaster muscle hemodynamics due to disruption of the deferential feed vessels.

Surgical preparation of the cremaster muscle for microvascular studies typically requires disruption of collateral vessels within the muscle and between the cremaster and the structures of the epididymis/ductus deferens. To study the effect of interrupting these vascular connections on cremaster hemodynamics, two modified preparations were examined in addition to the conventional open cremaster muscle preparation. One of these preparations enabled the measurement of feed vessel (1A) pressure and diameter with all cremaster vascular connections intact. The second preparation involved interruption of intramuscle collaterals to open the cremaster sac but with intact collateral pathways between the cremaster and deferential vessels. Intravascular pressures in the main cremasteric arteriole (1A) were similar in all three preparations with pressure in the 1A, expressed as a percentage of femoral artery pressure, varying between 53% in the intact preparation and 48% in the standard open preparation. These data support the existence of substantial upstream vascular resistance regardless of the extent of surgery. Selective occlusion of the branches of the deferential vessels significantly increased red cell velocity in the cremasteric 1A and major draining venule (1V) so that calculated blood flow increased by approximately 40% (P less than 0.01) in the 1A and 95% (P less than 0.01) in the 1V. Also, intravascular pressure fell significantly (P less than 0.01) in the 1A and increased in the 1V. Despite these compensatory changes total blood flow to the muscle was reduced by approximately 40% in the standard open preparation, compared to the preparation with the deferential feed pathway intact. Further studies where the 1A flow was transiently occluded indicated that the deferential pathway was capable of providing significant collateral blood flow to the muscle. Collectively these studies demonstrate that the surgical modifications of the cremaster vascular supply required for in vivo microscopy significantly alter normal hemodynamics within the vascular bed. The surgery does not, however, entirely explain the large pressure drop that exists upstream of the cremasteric 1A.

Responses of sequentially branching macro- and microvessels during reactive hyperemia in skeletal muscle☆

Small artery and microvascular responses during reactive hyperemia were compared to determine which resistance-bearing vessels played a role in controlling blood flow and resistance for the cremaster skeletal muscle. Using an intravital video microscopy system, measurements of microvessel pressure, flow velocity, and diameter were obtained from cremaster muscles in anesthetized rats. These were compared with measurements of diameter that were obtained from the small arteries feeding the cremaster muscle. After a 60-sec occlusion of the sacral aorta, total cremaster blood flow increased approximately 28% and calculated microvascular resistance for the cremaster muscle fell 50%. During the period of occlusion, diameters of small arteries (159-292 micron) decreased despite the presence of smooth muscle tone. Likewise, the diameters of large arterioles (65-117 micron) decreased whereas small arterioles (16-30 micron) dilated. The decrease in diameter of the small arteries and large arterioles was accompanied by a significant fall in intravascular pressure, suggesting that the behavior of these vessels was largely passive. Immediately following the release of occlusion, small arteries and large arterioles returned to their control diameters while small arterioles remained in a dilated state for approximately 2 min. These results indicate that for the cremaster muscle, vascular responses vary along the length of the arterial tree during reactive hyperemia, small but not large arterioles are primarily responsible for the decrease in network resistance and subsequent hyperemia following occlusion, and the small feeder arteries did not dilate during reactive hyperemia but instead acted to set a limit on the decrease in network resistance and the increase in blood flow.

Enhancement of histamine-induced vascular leakage by pertussis toxin in SJL/J mice but not BALB/c mice

Pertussis toxin (PTX) from Bordetella pertussis is known to enhance inflammatory responses which involve histamine and serotonin, including cell-mediated delayed-type hypersensitivity reactions. In this study we examined the effects of PTX on histamine-modulated microvascular responses. The actions of histamine on arteriole diameter and post-capillary leaky site formation in the cremaster muscle were measured intra-vitally in two inbred strains of mice (viz. BALB/c and SLJ). In SJL mice the rate and extent of histamine-induced leaky site formation were greatly enhanced (from 8.3 to 21.0 leaky sites per 0.1 cm2) by pre-exposure to PTX. In sharp contrast, PTX did not alter histamine-induced leaky site formation in BALB/c mice. Histamine-mediated dilation in arterioles in both strains of mice were not enhanced by PTX. PTX may enhance the development of inflammatory responses by enhancing histamine-induced leaky site formation of the microvasculature.

Use of surface-enhanced Raman spectroscopy for the detection of human integrins.

Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, alphaVbeta3 and alpha5beta1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.

Endothelin Mediates a Component of the Enhanced Myogenic Responsiveness Of Arterioles From Hypertensive Rats

Objective: To determine if the enhanced pressure‐induced constriction of arterioles isolated from hypertensive rats is mediated by the endothelium.