Immunohistochemical Identification of Arteriolar Development Using Markers of Smooth Muscle Differentiation

Arteriolar growth is an important event in the adaptation of normal tissues as well as in important pathologies, but the site of origin of new arterioles remains unknown. The network pattern of arteriolar development in skeletal muscle was detected by use of a new immunohistochemical technique that is based on the observation that fully differentiated (mature) vascular smooth muscle (SM) cells express both SM ax-actin and the two myosin heavy chains (MHCs) SM-1 and SM-2, whereas less differentiated (immature) vascular SM cells do not express MHC. The anterior gracilis muscle microvasculatures of 4and 9-week-old Sprague-Dawley rats were labeled with monoclonal antibodies to SM a-actin and to SM MHC. Whole transverse arteriole networks were observed, and terminal arterioles, defined as terminal segments labeled with SM a-actin, were classified on the basis of the presence or absence of SM MHC. A significantly different percentage of terminal arteriolar endings per network without SM MHC was observed in the two groups (66.1 + 17.3% for 4 weeks and 27.1 ± 18.5% for 9 weeks), suggest4 rteriolar growth is an important event in the j...g adaptation of normal tissues, such as exercised L XL. skeletal muscle1 and trained myocardium,2 as well as in important pathologies, including arterial collateralization following coronary artery stenosis,3 wound healing,4,5 and tumor growth.6,7 In skeletal muscle, new arteriolar growth is stimulated by exercise,1,8,9 ligation of the muscle's feed artery,10 and normal maturation.11 The site of origin of new arterioles within the microvascular network, however, remains unknown. Although it is widely accepted that capillaries grow by sprouting into the interstitium,612 an angiogenic sprouting process has not been observed in arterioles. The current hypothesis for arteriolar development is that certain capillaries, selected by presently unknown mechanisms, become invested with vascular smooth muscle (SM) cells and are therefore transformed into arterioles, a process denoted here as arterialization. Nehls and Drenckhahn'3 have suggested that pericytes, cells of mesenchymal origin located on the abluminal Received December 22, 1993; accepted May 9, 1994. From the Department of Biomedical Engineering (R.J.P., T.C.S.) and the Department of Molecular Physiology and Biological Physics (G.K.O.), University of Virginia, Charlottesville. Correspondence to Dr Thomas C. Skalak, Department of Biomedical Engineering, Box 377 Health Sciences Center, University of Virginia, Charlottesville, VA 22908. © 1994 American Heart Association, Inc. ing that arteriolar development is more nearly complete in the older animals. Sparsely distributed capillaries exhibited thin extensions of SM a-actin that crossed collecting venules and joined similar extensions from an adjacent transverse arteriole, effectively forming the basis for new arcade arterioles. SM a-actin and SM MHC labeling in terminal arterioles was always continuous with upstream arterioles. The pattern of labeling provides evidence that arterialization proceeds along capilaries from the terminal arteriolar endings, as opposed to being initiated at random sites on the developing vasculature that are distal to the arterioles. The methods described herein provide a powerful means to assess how factors such as tissue hypoxia, growth factors, and hemodynamic stresses affect arteriolar development as well as remodeling. (Circ Res. 1994;75:520-527.)