Duling, Brian R.
PO Box 800736
Cell-cell signaling in the Microvascular Wall
Our laboratory focuses on integrative biology of the vascular system, with two broad areas of excellence. First, we are interested in the cellular and molecular basis of the regulation of arteriolar tone and blood flow, especially in striated muscle. Second, we wish to understand the factors that control tissue oxygenation, and particularly the ways in which red cells are distributed among the microvessels.
Regulation of arteriolar tone
The essence of cardiovascular function is cellular coordination, and much of cardiovascular disease arises when coordination fails. The cardiac myocytes must be functionally linked together to form a pumping chamber, and the key molecules providing this linkage are the connexins, which form gap junctions in the intercalated disks. Less well known is the fact that gap junctions also integrate functions of the cells of the vessel wall (smooth muscle and endothelium). The resultant cell-cell communication modulates contraction, signaling, and synthetic processes - the gap junctions form a syncytium, the smooth muscle - endothelial cell unit.
Using intravital video microscopy in combination with electrophysiological and imaging techniques we have shown that vascular responses to stimuli are greatly influenced by the presence of the connexins. However, until recently the molecular basis for this communication, and its overall importance to cardiovascular health and disease could only be imagined. Now with the advent of molecular genetic approaches using homologous recombination, we have been able to delete specific connexin genes and even to delete selected genes in specific cells. Animals can now be produced with deletions of any of the vascular connexins, and for connexin 43 we can delete the connexin 43 gene in either smooth muscle or endothelium. These animals can be mated to produce multiple knockouts.
The physiological consequences of the connexin deletions are only beginning to be appreciated. Deletion of a connexin can result in either hypertension or hypotension depending on the cell in which the deletion occurs and the connexin that is deleted. The absence of appropriate intercellular signaling can lead to enormously exaggerated smooth muscle contraction, to fulminating hypertension, and cardiac hypertrophy. Perhaps most exciting, the connexins appear to be linked in a very complex regulatory scheme in which deletion of one connexin can cause a parallel reduction in a connexin in an adjacent cell, or of a different connexin in the same cell. There is virtually nothing known of the signaling and the regulatory processes that produce such coordinated regulation of connexin synthesis.
In addition to coordination of smooth muscle and endothelium, the connexins appear to play a role in the interactions between endothelium and leukocytes, and thus the connexins may be involved in the speed of attachment and emigration of white cells in inflammatory states. Current projects in the laboratory focus especially on the effects of connexin 40 deletions on electrically induced vascular wall signaling, on the modulation of catecholamine activation of smooth muscle by mural connexins, and on the role of afferent arteriolar connexins in the pathogenesis of hypertension.
A primary function of the vasculature is to regulate the concentration of oxygen in the tissues. Though essential, O2 is also toxic, and thus oxygen must be regulated within a narrow range, not merely provided in adequate amounts. This laboratory has used a variety of techniques to examine the patterns of and limits to tissue oxygenation, as well as the mechanisms of regulation of oxygen delivery. The amount of oxygen that can be delivered to the tissues is highly dependent on the hematocrit of blood in the capillaries, and we discovered that the capillary hematocrit is much lower than that of arterial blood, and that it is highly variable and subject to strict regulation. This discovery led to the realization that the variations in capillary hematocrit are due to variations in the thickness of a previously unrecognized intracapillary layer, the glycocalyx.
This layer is a typical extracellular matrix, which functions as the true interface between the blood and the endothelial cells. Electron microscopy shows that the glycocalyx is rather uniform over the surface of the endothelial cell, and that it is dynamic, being responsive to both hemodynamic forces and to inflammatory stimuli. At the present time, a major laboratory effort is underway to understand the molecular composition of the glycocalyx, the patterns of its synthesis, and the structural biology of the molecules that comprise it. Selective enzymatic digestion and capillary microperfusion have disclosed heparin and chondroitin sulfate, hyaluronic acid, albumin and globulins as part of a relatively stabile matrix that occupies more than half of the capillary cross-section. The laboratory is currently trying to understand the role played by the glycocalyx in regulating solute and water transport in the capillaries, and how it modulates leukocyte attachment and emigration.