The individual structural stages in capillary growth have been identified during development and under pathological circumstances in adults (wound healing, tumors), but there are no data to indicate whether these steps are similar when angiogenesis is induced in a fully differentiated microvascular bed in normal, uninjured adult skeletal muscle. In this study changes in capillary ultrastructure were correlated with capillary density and network morphology to elucidate the sequelae of angiogenesis in adult rat extensor digitorum longus (EDL) muscle whose activity was increased by stimulation at 10 Hz (8 h/day). This resulted in an increased capillary/fiber (C/F) ratio (based on staining for alkaline phosphatase) after 4 days; by 7 days C/F ratio was increased further, by approximately 50%. The ultrastructure of capillary endothelium in both the EDL and extensor hallucis proprius (EHP) was similar to control muscles after 2 days of stimulation, whereas endothelial cells in some capillaries in muscle stimulated for 4 days revealed signs of metabolic activation such as proliferation of organelles (Golgi apparatus, endoplasmic reticulum, ribosomes and mitochondria) and fewer pinocytic vesicles. Luminal surfaces were often irregular with numerous pseudopodial processes. Basement membranes were always present but amorphous regions were observed, particularly near pericyte processes. Unusually small capillary profiles, with either a slit-like lumen or with cisternae but no lumen, probably represented capillary sprouts. The interstitium contained increased collagenous and granular extracellular matrix surrounding capillaries, and numerous activated fibroblasts which were closely apposed to many capillaries. Capillary growth in EHP was also evaluated by confocal microscopy using whole mounts. The complex pattern of vessels underwent remodelling between 2 and 7 days of stimulation, resulting in more tortuous capillaries with numerous sprouts and loops. These combined observations suggest that angiogenesis may occur by a combination of sprouting, intussusceptive growth and elongation; also, that activation of endothelial cells occurs at the same time as disturbance of basement membranes during the earliest phase of growth and remodelling of the capillary bed. These changes are postulated to occur in connection with increased shear stress and/or capillary wall tension, which have been demonstrated previously.

To identify the sequence of events associated with the development of reduced vessel density (rarefaction) in hypertension, microvessel density and ultrastructure were assessed in the cremaster muscle of rats subjected to a 75% surgical reduction of renal mass and normotensive sham-operated control rats. Rats with reduced renal mass (RRM rats) and sham-operated rats were then maintained on either a high salt (4.0% NaCl) or a low salt (0.4% NaCl) diet for 3 days. Acute exposure to the high salt diet significantly increased mean arterial pressure in RRM rats but did not affect sham-operated control rats. Quantitative fluorescence microscopy of cremaster muscle whole mounts using rhodamine-labeled Griffonia simplicifolia I lectin revealed substantial rarefaction of microvessels in both RRM hypertensive rats and normotensive sham-operated rats on a high salt diet relative to corresponding control rats on a low salt diet. Confocal microscopy revealed a heterogeneous distribution of microvessels in RRM rats on a high salt diet, with some areas largely devoid of vessels. RRM and sham-operated rats on a high salt diet both exhibited changes in arteriolar ultrastructure, which included a loss of basement membranes and a dissociation of the endothelial and smooth muscle components of the vascular wall, resulting in a loss of vessel integrity. These observations demonstrate that a rapid loss of microvessels can occur not only in rats with RRM hypertension but also in normotensive rats on a high salt diet. This loss of microvessels results from structural alterations, which differ from the degenerative processes associated with microvascular rarefaction in rats with chronic RRM hypertension.

Commonly used methods for assessing reductions in microvascular density (rarefaction) in hypertension detect only perfused microvessels. In the present study, samples of cremaster and spinotrapezius muscles were taken from rats with chronic (4-week) reduced renal mass hypertension and normotensive sham-operated control rats, as well as from 12-week-old spontaneously hypertensive rats and their normotensive Wistar-Kyoto control strain. Mean arterial pressure was 149±8 mm Hg in the rats with reduced renal mass hypertension, 114±7 mm Hg in sham-operated rats, 177±9 mm Hg in spontaneously hypertensive rats, and 95±4 mm Hg in Wistar-Kyoto rats. Muscle samples were incubated with rhodamine-labeled Griffonia simplicifolia I lectin, which identifies both perfused and nonperfused microvessels. Microvascular density was assessed by counting intersections with a 20-/um grid. Microvessel density was significantly reduced in cremaster muscles of both spontaneously hypertensive and reduced renal mass hypertensive rats, and in the spinotrapezius muscle of spontaneously hypertensive rats, compared with their respective normotensive controls. Further studies in the reduced renal mass rats on low salt diets indicated that lectin binding was also decreased as salt intake was increased, independent of blood pressure. This change was not due to an alteration in lectin-binding affinity. These studies indicate that lectin binding can be a useful tool for assessing microvessel density that does not depend on the perfusion state of the vessels and that rarefaction due to hypertension is not evenly distributed in all vascular beds. These results also provide evidence that dietary salt intake alone can influence microvessel density, as measured by the lectin technique. {Hypertension 1990;15:779-783) C onsiderable evidence has been accumulated to indicate that both changes in vessel caliber and vessel number can contribute to the increased vascular resistance in hypertension.

The cellular and subcellular responses related to the survival or destruction and subsequent regeneration of muscle fibers within the freely grafted extensor digitorum longus muscle of the rat were examined by light and electron microscopy. A small number of fibers at the periphery of the grafts survived the initial ischemia but underwent denervation changes and accumulated lipid deposits. The majority of fibers in the grafts, however, became ischemic and underwent an intrinsic degeneration within 4 hours. Cell-mediated destruction of the degenerating fibers occurred as the grafts became revascularized. The basal laminae and some of the satellite cells were the only elements of the original fibers that persisted. Regeneration began at the periphery of the graft within three days after grafting and reached the center about three days later. After phagocytosis of the original fibers, presumptive myoblasts within the grafts differentiated into myoblasts and myotubes. The formation of myotubes followed a biphasic pattern of development comparable to that of normal fetal muscle. Although most of the myotubes were formed within the basal lamina remaining from the original fiber, there was also evidence for regeneration outside the basal lamina. Myotubes matured into muscle fibers which were essentially normal in apperance when examined up to 180 days after grafting. Some fibers, however, were atrophic, presumably due to a failure to become innervated, and some fibers were joined by myo-myous junctions. Pre-denervated grafts and Marcaine-treated grafts were also examined. There were more surviving fibers in pre-denervated grafts, and cell-mediated destruction of degenerating fibers proceeded more rapidly than in normal grafts. No surviving fibers were found in Marcaine-treated grafts. The changes in these grafts were otherwise similar to normal grafts. A schematic model of the spatial and temporal sequence of degeneration and regeneration within a free muscle graft is presented.