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A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of "angiogens", mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore we sought to examine a novel method of stimulating myocardial angiogenesis through hypoxic preconditioning at both capillary (using anti-CD31) and arteriolar (using anti- alpha smooth muscle actin) levels and also investigate whether such treatments could preserve left ventricular contractile functional reserve and regional blood flow by increasing vascular endothelial growth factor (VEGF). Male Sprague-Dawley rats were randomly divided into four groups: normoxia+sham surgery (CS), normoxia+permanent left anterior descending coronary artery (LAD) occlusion (CMI), hypoxic preconditioning+sham surgery (HS) and hypoxic preconditioning+permanent LAD occlusion (HMI). Rats in the preconditioned groups were subjected to systemic hypoxemic hypoxic exposure (10+/-0.4% O(2)) for 4 h followed by a 24 h period of normoxic reoxygenation prior to undergoing LAD occlusion. Rats in the normoxia group were time matched with the preconditioned group and maintained under normoxic conditions for a 28 h period prior to LAD occlusion. Western blot analysis was performed to measure VEGF expression and TUNEL staining with endothelial cell-specific antibody, anti-VWF, was used to examine endothelial apoptosis. One, two and three weeks after the LAD occlusion, baseline left ventricular pressures were monitored and recorded. Pharmacological stress tests with dobutamine infusion in progressively increasing doses revealed significantly elevated contractile reserve at each dose point in the HMI group compared to the CMI group. The HMI group displayed statistically significant increases in capillary as well as arteriolar density after 1, 2 and 3 weeks post-operation. Blood flow was also significantly elevated in the HMI groups when compared to the CMI group. The extent of endothelial cell apoptosis was found to be inversely proportional to VEGF expression. It was concluded that hypoxic preconditioning stimulates myocardial angiogenesis to an extent sufficient to exert significant cardioprotection in a rat model of myocardial infarction progressing to heart failure as evidenced by increased capillary/arteriolar density and enhanced ventricular contractile functional reserve., (Copyright 2002 Academic Press.)

The murine VEGF gene is alternatively transcribed to yield the VEGF(120), VEGF(164), and VEGF(188) isoforms, which differ in their potential to bind to heparan sulfate and neuropilin-1 and to stimulate endothelial growth. Here, their role in retinal vascular development was studied in mice selectively expressing single isoforms. VEGF(164/164) mice were normal, healthy, and had normal retinal angiogenesis. In contrast, VEGF(120/120) mice exhibited severe defects in vascular outgrowth and patterning, whereas VEGF(188/188) mice displayed normal venular outgrowth but impaired arterial development. It is noteworthy that neuropilin-1, a receptor for VEGF(164), was predominantly expressed in retinal arterioles. These findings reveal distinct roles of the various VEGF isoforms in vascular patterning and arterial development in the retina.

Arteriolar growth accompanying capillary angiogenesis has been linked with hemodynamic factors resulting from increased blood flow. Here we describe the growth of arterioles occurring in rat skeletal muscles stretched by an overload due to the removal of agonist muscles, where blood flow was not increased, and we provide morphological evidence for the type of cells involved in this growth. Rat extensor digitorum longus (EDL) and extensor hallucis proprius (EHP) were overloaded by unilateral extirpation of their agonist, tibialis anterior. EDL muscles were taken for immunohistochemistry in cryostat sections to mark endothelial cells (Griffonia simplicifolia I, GSI lectin), smooth muscle cells and pericytes (alpha smooth muscle actin, alphaSMA), and "mature" arterioles (smooth muscle myosin heavy chains). EHP muscles were used for corresponding evaluation by confocal and electron microscopy. The number of capillaries surrounding muscle fibers was not significantly different after 1 week of stretch but was higher after 2 weeks (5.15 +/- 0.2 vs 4.3 +/- 0.2 in controls, P < 0.05). Similarly, capillary density (CD) and capillary/fiber ratio (C/F) gradually increased (CD 778 +/- 86 at 2 weeks vs 593 +/- 35 mm(-2) in controls, C/F 2.07 +/- 0.13 vs 1.38 +/- 0.06, respectively). In contrast, the number of alphaSMA-positive vessels around fibers increased after 1 week (2.16 +/- 0.09 vs 0.25 +/- 0.02 in controls) and was lower after 2 weeks (1.42 +/- 0.24, P < 0.05, vs 1 week). Arteriolar density was higher at 1 (110.9 +/- 7.5 mm(-2)) and 2 weeks (70.7 +/- 12.1) with respect to controls (31.0 +/- 1.6 mm(-2)). The increased density was greater in alphaSMA-positive vessels <10 microm in diameter (controls 18.0 +/- 1.04, 1 week 77.2 +/- 4.5, 2 wk 42.2 +/- 9.0 mm(-2)) than in vessels >10 microm (13.0 +/- 0.8, 33.7 +/- 4.0, 29.5 +/- 4.7 mm(-2)). Electron microscopy showed "activated" (TEM fine structure) and proliferating (immunogold labeling for BrdU) fibroblasts in the vicinity of capillaries, some of which were embedded in the capillary basement membrane, consistent with a transformation into pericytes and possibly later smooth muscle cells. Confocal microscopy indicated that some mesenchymal cells became GSI positive and formed extended processes which contacted capillaries via tapered endings. Growth of arterioles in stretched muscles appears to involve proliferation of fibroblasts, which may migrate toward capillaries and precedes any apparent increase in capillarization., (Copyright 2001 Academic Press.)

Using intravital fluorescence microscopy in the ears of hairless mice, we determined skin microvascular adaptations during the process of aging from juvenile to adult and senescent life (6-78 wk). Despite an increase of ear area within the first 36 wk, the number and branching pattern of both arteriolar and venular microvessels remained constant during the whole life period. Both arterioles and venules exhibited an increase in length, diameter, and intervascular distance up to the age of 36 wk. With the increase of the size of the ears, the observation that cutaneous capillary density remained unchanged implied new capillary formation. During aging to 78 wk, capillary density in the ears was reduced to approximately 40%. Functional analysis revealed an appropriate hyperemic response to a 2-min period of ischemia during late juvenile and adult life, which, however, was markedly reduced during senescence. Thus, except for capillaries, there is no indication for age-related new vessel formation. The process of aging from adult to senescent life does not cause any significant remodeling but is associated with a decrease of nutritive perfusion and a functional impairment to respond to stimuli such as ischemia.

Tissue specimens of nine normal human pancreata from fetuses, neonates, and adults were subjected to serial sectioning and computer-assisted three-dimensional (3-D) reconstruction in an effort to study the growth of Langerhans' islets based on their distribution in the lobule and their relationship with the ducts and arterioles. Also, islet volumes were obtained by 3-D morphometry. In 24-week-old fetuses, the islets were shown to be in direct contact with the ducts, whereas in the neonates, they gradually became separated from the ducts, finally dispersing throughout the lobule in the adults. This transition seemed to allow islet hormones to have physiological effects on the exocrine function of the acinar tissue, making the pancreas achieve the endocrine-exocrine correlation. However, half of the islets remained next to the ducts even in the adult pancreata. With regard to the relationship between the islets and arterioles, "arteriolar" islets having an afferent arteriole accounted for approximately 20% in number, but their volume cumulated to approximately 70% of the total, comprising a greater part of the pancreatic endocrine gland.

Ageing in mammals is characterized by a decline in plasma levels of insulin-like growth factor-1 that appears to contribute to both structural and functional changes in a number of tissues. Although insulin-like growth factor-1 has been shown to provide trophic support for neurons and administration of insulin-like growth factor-1 to ageing animals reverses some aspects of brain ageing, age-related changes in insulin-like growth factor-1 or type 1 insulin-like growth factor receptors in brain have not been well documented. In this series of studies, insulin-like growth factor-1 messenger RNA and protein concentrations, and type 1 insulin-like growth factor receptor levels were analysed in young (three to four- and 10-12-month-old), middle-aged (19-20-month-old) and old (29-32-month-old) Fisher 344 x Brown Norway rats. Localization of insulin-like growth factor-1 messenger RNA throughout the lifespan revealed that expression was greatest in arteries, arterioles, and arteriolar anastomoses with greater than 80% of these vessels producing insulin-like growth factor-1 messenger RNA. High levels of expression were also noted in the meninges. No age-related changes were detected by either in situ hybridization or quantitative dot blot analysis of cortical tissue. However, analysis of insulin-like growth factor-1 protein levels in cortex analysed after saline perfusion indicated a 36.5% decrease between 11 and 32 months-of-age (P<0.05). Similarly, analysis of type 1 insulin-like growth factor receptor messenger RNA revealed no changes with age but levels of type 1 insulin-like growth factor receptors indicated a substantial decrease with age (31% in hippocampus and 20.8 and 27.3% in cortical layers II/III and V/VI, respectively). Our results indicate that (i) vasculature and meninges are an important source of insulin-like growth factor-1 for the brain and that expression continues throughout life, (ii) there are no changes in insulin-like growth factor-1 gene expression with age but insulin-like growth factor-1 protein levels decrease suggesting that translational deficiencies or deficits in the transport of insulin-like growth factor-1 through the blood-brain barrier contribute to the decline in brain insulin-like growth factor-1 with age, and (iii) type 1 insulin-like growth factor receptor messenger RNA is unchanged with age but type 1 insulin-like growth factor receptors decrease in several brain regions. We conclude that significant perturbations occur in the insulin-like growth factor-1 axis with age. Since other studies suggest that i.c.v. administration of insulin-like growth factor-1 reverses functional and cognitive deficiencies with age, alterations within the insulin-like growth factor-1 axis may be an important contributing factor in brain ageing.

Objective: The purpose of this study was to test the hypothesis that capillary growth induced by chronic electrical stimulation of skeletal muscle is accompanied by the growth of small arterioles., Methods: Lower limb flexor muscles of Sprague-Dawley rats were stimulated by electrodes implanted in the vicinity of the peroneal nerve at 10 Hz for 8 h/d for 2 and 7 days. Cryostat sections from the proximal, middle, and distal regions of the extensor digitorum longus muscle (EDL) were fluorescently immunolabeled with alpha-smooth muscle actin (alpha SMA) and myosin heavy chain (MHC) to identify mature (alpha SMA and MHC-positive) and immature (alpha SMA-positive, MHC-negative) arterioles. The fluorescent derivative of the lectin Griffonia simplicifolia I (GSI) was used to identify all microvessels, including arterioles, capillaries, and venules., Results: The number of vessels positive for GSI or alpha SMA surrounding muscle fibers was similar in all three muscle regions (proximal, middle, distal). The mean values +/- SEM for GSI-positive vessels from all regions were similar in control (4.3 +/- 0.07) and 2-day stimulated (4.7 +/- 0.08) but higher in 7-day stimulated muscles (6.7 +/- 0.1, p < 0.05), thus confirming the previous findings on capillary growth. A similar increase was found in the number of alpha SMA positive vessels < or = 10 microns outer diameter (1.3 +/- 0.09 versus 0.4 +/- 0.03 around muscle fibers in controls). The density of terminal arterioles (< or = 10 microns) was slightly but not significantly higher after 2 days of stimulation (19.5 +/- 4 versus 15.6 +/- 2 profiles/mm2 in control muscles) and significantly higher after 7 days (33 +/- 7). While a similar increase was observed in the density of preterminal arterioles > 10 microns (17 +/- 3 control, 22 +/- 3 at 2 days and 40 +/- 5 at 7 days), the density of MHC-positive vessels muscles stimulated for 7 days was unchanged. Seven-day stimulated muscle also had a fivefold higher density of microvessel profiles < or = 10 microns that were only partially surrounded by alpha SMA. This considerably exceeds the relative increase in the number of capillaries and thus supports the concept of arteriolar growth by transformation from capillaries., Conclusions: Chronic electrical stimulation results in an early increase in the number of immature (MHG-negative), but not mature (MHC-positive) arterioles, a process that accompanies the increase in capillarization. The great increase in the number of microvessels only partially covered by alpha SMA suggests arteriolization of capillaries as a contributing mechanism in this growth.

Objective: To investigate the spatial and phenotypic origin of the new smooth muscle (SM) cells that are necessary for transverse arteriolar (TA) remodeling by establishing the distribution of cellular proliferation in TA trees during maturation., Methods: Whole-mount gracilis muscles from rats at 4 and 9 weeks of age were immunolabeled for SM myosin heavy chain to denote arterioles and for bromodeoxyuridine to denote S-phase (DNA synthesizing) nuclei. The dimensions of each clearly visible segment in TA trees were measured. S-phase cells in the wall of, or within 5 microns of, TA segments were identified as (1) endothelium or intimal fibroblasts, (2) SM, or (3) interstitial cells. The relative percentages of each cell type in S-phase, the distribution of arteriolar diameters containing S-phase SM, and the density of S-phase interstitial cells (per unit length and per unit surface area of TA) were determined. Alcian blue counterstaining was used to discern the percentage of interstitial cells that were mast cells., Results: At 4 and 9 weeks, respectively, 3.7% and 2.1% of S-phase cells were endothelium or intimal fibroblasts, 3.0% and 4.2% were SM, and 93.3% and 93.7% were interstitial cells. No S-phase interstitial cells within 5 microns of TAs were mast cells. The mean diameter of TA segments containing as S-phase SM nucleus was 15.22 +/- 1.2 microns at 4 weeks of age, with the minimum diameter being 8.9 microns. From 4 to 9 weeks of age, the number of interstitial cells per unit length of TA decreased 10-fold from 15.2 (n = 115) to 1.5 (n = 182) cells/min. At 4 weeks, the density of S-phase interstitial cells was greatest surrounding the most terminal arterioles., Conclusions: When coupled with the result that S-phase SM is absent in the most terminal segments, the relatively high density of S-phase interstitial cells surroundings the smallest diameter terminal segments at 4 weeks of age is consistent with the hypothesis that fibroblast hyperplasia is a component of terminal arteriolar development.

To define the relationship between renal vascular development and renin distribution during kidney ontogeny, the complete renal arterial tree of Sprague Dawley rats during fetal (20 d) and postnatal (1 to 90 d) life was microdissected and immunostained for renin. A shift in renin distribution from interlobar and arcuate arteries in the fetus to the afferent arterioles in the adult was observed. In addition, seven types of renin distribution along the afferent arterioles were identified. In type I, renin was distributed continuously along the whole length of the afferent vessel. This pattern was most frequently observed in the fetus. In type II, renin extended upstream from the glomerulus but did not occupy the whole length of the arteriole. This type was relatively constant throughout postnatal life. In type III, renin was present as bands along the afferent vessel; it was most frequently observed in the fetal and early perinatal periods. In type IV, renin was restricted to the "classical" juxtaglomerular localization. It was the most frequent type observed in the adult rat. In type V, no renin was found in the arteriole. It was the second most frequent type observed in the adult rat. In addition, two "mixed" patterns, type III/IV and type III/II, were occasionally observed. The distribution of renin-expressing cells was spatially and temporally associated with the development of blood vessels. Development of a new arterial branch was preceded by the appearance of renin-expressing cells at the point of branching. This was followed by an outpouching of the arterial wall that progressively elongated to form a new arteriole. During this process, renin-expressing cells were distributed along the whole of the newly formed vessel. As the vessel matured, renin-expressing cells became restricted to the juxtaglomerular portion of the afferent arteriole. It is concluded that throughout life and within each individual arterial tree, expression of renin is heterogeneous, following patterns that are unique for each developmental stage. Furthermore, the association of renin-expressing cells with branching of renal arterioles suggests a role for these cells in the development of the kidney vasculature.

The differentiating cavernous body (CB) of postnatal rabbit penises was examined with a special reference to immunolocalizations for fibronectin (FN) and endothelin-1 (ET-1). At postnatal day 1, the CBs were embedded by an abundance of mesenchymal cells (MCs), and some of them were closely associated with endothelial cells of preexisting capillaries. Our electron micrographs indicated that such MCs are successively incorporated into the capillary endothelium as vasoformative cells. At this period, vascular sprouts of the helicine artery (HA), which were associated with the MCs, arose from the deep penile artery, and the transformation of such cells to endothelial and medial muscle ones was also indicated, and some MCs appeared to differentiate to epithelioid cells in the media. Immunoreactions for FN were preferentially localized in the rough endoplasmic reticulum (rER) and along the plasma membrane of such vasoformative MCs, and on the extracellular matrix components which connect these MCs with sprouts of both growing capillaries and HA. These findings suggest that FN, which is produced in the rER of the MCs, plays a crucial role in the mechanical linkage during the incorporation of vasoformative MCs into these penile vessels. Immunoreactions for ET-1 were preferentially localized on Weibel-Palade bodies in endothelial cells of the HA, implying the involvement of this peptide in the regulation of the local blood flow in this vessel.

Several reports have demonstrated that cerebral blood flow decreases with age and may contribute to neurodegenerative changes found in aging animals and man. Because GH and insulin-like growth factor 1 (IGF-1) decrease with age and have an important role in vascular maintenance and remodeling, we hypothesized that the decrease in cerebral blood flow is associated with a rarefaction of cerebral blood vessels resulting from a decline in GH and IGF-1. Measurements of vascular density (number of vessels/cortical surface area) in both Brown-Norway and Fisher 344/Brown-Norway rats were made at 5, 13, and 29 months of age using chronic cranial window chambers that allowed viewing of the cortical surface and its corresponding vasculature. Correlations were made with plasma levels of IGF-1. In Brown-Norway rats, arteriolar density decreased from 15.53 +/- 1.08 to 9.49 +/- 0.62 endpoints/mm2 in 7- and 29-month-old animals, respectively (P < 0.05). A decline was observed also in arteriolar anastomoses [3.05 +/- 0.21 to 1.42 +/- 0.24 connections/mm2 in 7- and 29-month-old animals (P < 0.05)]. Venular density did not decrease with age. Similar changes were observed in Fisher 344/Brown-Norway rats. The number of cortical surface arterioles was correlated with plasma IGF-1 levels at the time of vascular mapping (r = 0.772, P < 0.05), and injection of bovine GH (0.25 mg/kg, s.c., twice daily for 35 days) to 30-month-old animals increased both plasma IGF-1 and the number of cortical arterioles. These data indicate that: 1) vascular density on the surface of the cortex decreases with age; 2) vascular density is correlated with plasma levels of IGF-1; and 3) injection of GH increases cortical vascular density in older animals. We conclude that GH and IGF-1 have an important role in the decline in vascular density with age and suggest that decreases in vascular density may have important implications for the age-related decline in cerebral blood flow and brain function.

Cardiopulmonary bypass (CPB) is associated with a generalized defect in the intrinsic control of vascular smooth muscle. To determine if myogenic reactivity of skeletal muscle arterioles was altered by CPB, sheep (n = 7) were placed on hypothermic CPB (27 degrees C) for 90 min and hearts were arrested by cold blood cardioplegia ([K+] = 25 mM) for 60 min. Arterioles (70-180 microns) were isolated from the gracilis muscle before (control) and 15 min after CPB. In vitro arteriolar responses were studied with video-microscopy. Myogenic reactivity was examined to stepwise increases in intraluminal pressure from 10 to 100 mm Hg. Mean arterial pressure was decreased from 80 +/- 15 prior to CPB to 55 +/- 4 mm Hg (P < 0.01) 15 min after CPB. Myogenic contraction was observed in control vessels and was markedly attenuated by the protein kinase C inhibitor staurosporine (P < 0.01). CPB decreased myogenic contraction and shifted the pressure-diameter relation upward, suggesting a decrease in the intrinsic tone (both P < 0.05 vs control). CPB reduced contractile responses to the alpha 1-adrenoceptor agonist phenylephrine from -43 +/- 7% to -23 +/- 5% (P < 0.01) and the protein kinase C activator 12-deoxyphorbol 13-isobutyrate 20-acetate (phorbol ester) from -64 +/- 6% to -38 +/- 16% (P < 0.01). CPB-associated decrease in myogenic reactivity of skeletal muscle arterioles is likely due to alterations in protein kinase C and/or downstream signal transduction. This may account in part for reduction in systemic vascular resistance and hypotension associated with CPB.

To better understand normal microvascular network growth, we conducted a longitudinal study on the spinotrapezius muscle of Sprague-Dawley rats of 3 ages: weanling (3-4 weeks), juvenile (7-8 weeks) and mature (11-12 weeks). Morphometric analysis revealed that despite dramatic growth of the muscle (from 117 +/- 60 mg in weanling to 417 +/- 112 mg in mature rats), there is no significant change in the total number of arcade arteriole segments per network over this period (from 140 +/- 35 to 181 +/- 51). The mean arcade segment length increased over this period (from 0.96 +/- 0.17 to 1.45 +/- 0.32 mm), but not in proportion to tissue growth. Consequently, the total arcade segment length per unit muscle volume significantly decreased (from 1.36 +/- 0.48 to 0.66 +/- 0.12 mm/mm3). The estimated number of transverse arteriolar trees per muscle (approximately 600) did not appreciably change with growth, leading to a decrease in the number of trees per millimeter arcade arteriole (from 4.4 +/- 1.2 to 2.3 +/- 0.6). Transverse arteriolar trees underwent age-dependent increases in the number of segments within each branch order and in mean segment length. These observations suggest that arteriolar network growth during juvenile maturation occurs by elongation of pre-existing arcade and transverse vessels with angiogenesis occurring in the distal segments of transverse arteriolar trees.

The arteriolar network undergoes structural adaptation in several physiological and pathological conditions, including exercise, maturation, hypertension, and reduced tissue perfusion due to arterial ligation. Although many physical and biochemical stimuli for arteriolar adaptation have been proposed, the individual contributions of these specific stimuli have yet to be elucidated. We tested the hypothesis that hemodynamic stress is an important determinant of growth and remodeling in the arteriolar network. An immunofluorescence, dual-labeling technique for the smooth muscle (SM) contractile proteins SM alpha-actin and SM myosin heavy chain (MHC) was used to assess terminal and arcade arteriolar (AA) remodeling in the rat gracilis muscle arteriolar network in response to chronic vasodilation, a stimulus that elevates circumferential wall stress levels in the arterioles and capillaries. SM alpha-actin, a marker of SM from the earliest stages of differentiation, was used to delineate the terminal and AAs. SM-MHC, a marker of SM in later stages of differentiation, was used to assess the relative maturity state of SM in terminal arteriolar endings. Mean percentage of SM-MHC negative terminal arteriolar endings per muscle, a measure of terminal arteriolar development, increased from 37.6 to 56.0% after 1 wk of prazosin treatment and from 36.3 to 57.6% after 2 wk of treatment. Mean number of AA segments with diameters < 15 microns increased more than threefold from 1.25 to 5.25 after 2 wk, consistent with the formation of new AA segments by the anastomoses of small-diameter terminal arterioles. Because arteriolar remodeling proceeded in a network pattern that has been shown to be consistent with a circumferential wall stress-growth rule and inconsistent with a wall shear stress-growth rule, the experimental results suggest that circumferential wall stress is a stimulus for arteriolar network remodeling.

This study was designed to determine the effects of angiotensin II infusion on structure of conduit and resistance arteries and to see if the effects correlate with changes in platelet-derived growth factor A chain (PDGF-A) gene and protein expression. Wistar rats were subcutaneously infused by osmotic minipump with either angiotensin II (ANG II) at 200 ng.kg-1.min-1 or physiological saline (control) for 14 days. Tail-cuff systolic blood pressure was significantly higher in ANG II compared with control rats beginning the second day of infusion and continuing to the end of 2 wk. Both aorta and external spermatic artery (first-order arteriole of the cremaster muscle) developed increased wall-to-lumen ratios in the ANG II rats, but this occurred by hypertrophy of the wall in the aorta and reduction of the lumen in the arteriole. Digoxigenin-labeled cRNA probes were used for in situ hybridization of vascular sections to identify PDGF-A mRNA. Gene expression of PDGF-A in ANG II rats was upregulated in the hypertrophied aorta and the nonhypertrophied arteriole. With the use of immunocytochemistry techniques, PDGF-A and proliferating cell nuclear antigen were increased in the aorta but not in the arterioles of ANG II rats compared with control rats. These results suggest that the difference in growth response between the aorta and the arteriole induced by ANG II may lie in posttranscriptional modification of PDGF-A mRNA, differential control of transition, or turnover of PDGF-A protein.

It has recently been shown that following treatment with colony-stimulating factor-1 (CSF-1) the osteopetrotic condition in toothless (tl) rats greatly improves and growth is accelerated. We have examined the effects of such treatment on the microvasculature of the distal femoral chondro-osseous junction, a site where bone growth in length is coordinated with angiogenesis. Vascular casts and ultrastructural analyses of this region showed that, compared to untreated normal rats, untreated mutants showed little bone growth or angiogenesis. When mutants were treated with CSF-1 angiogenesis was markedly accelerated. These data show a remarkable effect of this growth factor on angiogenesis in this osteopetrotic mutation. Whether this effect of CSF-1 on angiogenesis is direct or indirect is not known and indicates that its effects on the normal microvasculature deserve further study.

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 alpha-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 4- and 9-week-old Sprague-Dawley rats were labeled with monoclonal antibodies to SM alpha-actin and to SM MHC. Whole transverse arteriole networks were observed, and terminal arterioles, defined as terminal segments labeled with SM alpha-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), suggesting that arteriolar development is more nearly complete in the older animals. Sparsely distributed capillaries exhibited thin extensions of SM alpha-actin that crossed collecting venules and joined similar extensions from an adjacent transverse arteriole, effectively forming the basis for new arcade arterioles. SM alpha-actin and SM MHC labeling in terminal arterioles was always continuous with upstream arterioles.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether the alterations in coronary vascular resistance with aging have a structural basis, the quantitative properties of the intramural branches of the coronary circulation and capillary network were measured in Fischer 344 rats at 4, 12, 20, and 29 mo. Physiological measurements demonstrated that a severe impairment in cardiac pump function developed with age, leading to the occurrence of ventricular failure at 29 mo. Morphometrically, the length densities of resistance vessels from 6 to 20 microns in luminal diameter markedly decreased at 12 mo, and this change persisted at 20 and 29 mo. This phenomenon affected the inner, middle, and outer layers of the left ventricular wall. In contrast, capillary luminal volume percentage, capillary numerical density, and average diffusion distance for oxygen in the left and right myocardium were altered only at 20 mo, whereas values comparable to those at 4 mo were found at 12 and 29 mo. The preservation of these capillary characteristics in the heart was due to a significant amount of capillary proliferation. In conclusion, aging effects lead to rarefaction of coronary arterioles in the myocardium, which may impair coronary resistance and reserve without altering the capillary microvasculature and the oxygenation potential of the old and senescent heart.

The first goal of this study was to examine the hypothesis that aging impairs dilator responses of cerebral arterioles to N-methyl-D-aspartate (NMDA). The second goal was to determine whether aging impairs vasodilatation in response to activation of ATP-sensitive K+ channels. Diameter of cerebral arterioles was measured through a cranial window in anesthetized adult (5 +/- 1 (mean +/- S.E.) months old) and old (26 +/- 1 months old) Fischer 344 rats. In adult rats, NMDA (10 and 100 microM) dilated pial arterioles by 14 +/- 5 and 72 +/- 9%, respectively, from a control diameter of 36 +/- 5 microns. Aprikalim (1 and 10 microM), a direct activator of ATP-sensitive potassium channels, dilated cerebral arterioles in adult rats by 14 +/- 3 and 49 +/- 6%, respectively. Vasodilatation in response to NMDA and aprikalim were similar in old and adult rats. Thus, in contrast to impaired cerebral vasodilator responses to some stimuli, responses of cerebral arterioles to NMDA and activation of ATP-sensitive potassium channels are preserved in old Fischer 344 rats.

Quantitative changes in the terminal vascular bed of the mammalian heart were assessed during postnatal development and aging. The most striking feature is a considerable formation of new capillaries in the early postnatal period, accompanied by a moderate formation of new arterioles. On the other hand, coronary arterioles seem to disappear at a higher rate than capillaries in the senescent heart. We proposed a three-dimensional structural model of tissue capillary supply, defined as capillary domain area times capillary segment length. This so called capillary supply unit increases as a function of age and body growth. It is very similar in size and shape (length to width ratio) to cardiac myocytes.

The objective of this study was to evaluate the role of nitric oxide (NO) in the regulation of whole kidney and glomerular hemodynamics during aging. After 2 wk of oral treatment with N-nitro-L-arginine methyl ester (L-NAME; 4.5 mg.kg body wt-1.day-1) to inhibit NO synthesis, male rats, aged 3-5, 13-15, and 21-24 mo, were studied by micropuncture. Blood pressure increased by 50% in old (21-24 mo) rats with L-NAME but only 20-30% in the two younger groups. With L-NAME, renal vascular resistance increased fivefold in old rats but only twofold in younger groups. Glomerular capillary pressure increased 20-30% in younger L-NAME rats and 60% in older rats. Afferent and efferent resistances increased dramatically, and the glomerular capillary ultrafiltration coefficient decreased in all L-NAME-treated rats but most strikingly in the 21- to 24-mo-old group. Acute infusion of L-arginine significantly attenuated the effects of NO synthase inhibition on arterial pressure and renal hemodynamics in both young and old rats. This study confirms that NO synthesis blockade has a greater effect on renal hemodynamics in aging rats and implies that NO may play a progressively more important role in controlling renal function with advancing age.

Coronary reserve has been shown repeatedly to be depressed in hypertension and aging. The underlying mechanisms remain elusive, but structural alterations of the coronary vasculature have been implicated. In this study, we measured maximal coronary dilator capacity and structural characteristics relevant to coronary resistance in aging normotensive (Wistar-Kyoto, n = 22) and spontaneously hypertensive rat (SHR) strains (n = 25) at 1.5, 4, 11, 16, and 22 months of age. Coronary flow measurements, using radiolabeled microspheres, demonstrated a significant (p < 0.01) hypertension- and age-related decline in maximal coronary dilator capacity. After flow measurements, vascular dimensions and arteriolar density were obtained from 1-micron sections prepared from perfusion-fixed hearts. A total of 10,012 arterioles were analyzed, 4,820 in hypertensive and 5,192 in normotensive rats. There was an 18-28% reduction in arteriolar density in hypertensive rats that specifically affected the terminal arteriolar bed at 1.5-11 months. However, the decrement in arteriolar density stabilized at 10% and 6% in 16- and 22-month-old hypertensive rats, respectively. Arteriolar density was not affected by aging. In both strains, there was a significant (p < 0.01) age-related decrease in the ratio of lumen diameter to wall thickness in arterioles > 50 microns. In addition, there was an overall 30% decrease (p < 0.01) in the ratio of lumen diameter to wall thickness in hypertensive compared with normotensive rats. These data indicate that both hypertension and aging are accompanied by structural alterations of the coronary resistance vasculature. These structural alterations may contribute to the depression in coronary reserve that complicates hypertension and aging.

Adaptive responses of mature arterioles were examined after a 38% reduction in total blood flow to the cremaster muscle produced by unilateral orchidectomy in 12-wk-old rats. Four weeks later, the muscle was smaller than the contralateral cremaster, which did not increase in size during this period. Measured by closed-circuit television microscopy, the internal diameters of first- through fourth-order arterioles (1A-4A) were smaller, but wall cross-sectional area was reduced only in 3As. The smaller diameter of the 1A in the orchidectomy muscle resulted in unchanged wall shear rate. As determined from the perfusion-fixed, microfilled cremaster muscles, the total length of the arcading arterioles and the number of 3As were not statistically different, but the total number of 4As was significantly reduced on the orchidectomy side. Therefore, chronic load reduction in a mature muscle resulted in reduced blood flow, decreased number of 4As, and smaller arteriolar internal diameters in the absence of net changes in vascular wall cross-sectional area. A local autoregulatory mechanism related to flow-induced shear stress is suggested as the mechanism mediating the changes.

To determine the effects of postnatal growth on the coronary microcirculation, the geometry of capillary nets was studied in male rats at 21, 28, 60, and 240 days of age. Tissue sections were exposed to a histochemical technique that distinguished arteriolar (AC) and venular (VC) capillary regions by color. The tissue area (capillary domain) surrounding individual capillaries progressively increased with growth of the heart (P less than 0.01). In all groups, AC domain area was larger than VC domain area (P less than 0.01). The heterogeneity of capillary spacing, which is another important determinant of oxygen supply, demonstrated significant increases with age for AC regions (P less than 0.01). With increasing left ventricular mass, there was a lateral expansion of the arteriovenous capillary set, which was displayed by an increase in the number of and distance between adjacent capillaries that traversed the distance from arteriole to venule (P less than 0.01). Our data indicate that differences in the geometry of arteriolar and VC regions are present by 21 days of age. During postnatal growth, the results from both cross and longitudinal sections suggest that the geometrical conditions for oxygen supply are notably reduced.

We compared six young (1 year) and six senescent (11 years) purebred beagles to determine the effects of aging on the coronary microvasculature. The hearts were perfusion-fixed in vitro, and myocardial specimens were subjected to microscopic image analysis. Absolute left ventricular mass increased by 55% with age, while cardiocyte cross-sectional area increased by 10% and 30% in the midmyocardium and endomyocardium, respectively. Although capillary numerical density was lower in the senescent dogs (16% in midmyocardium and 19% in endomyocardium), volume density was similar in the two groups because capillary diameter increased significantly with age in both regions. Capillary length density was reduced by 27% with age in the endomyocardium of the old beagles. Wall/lumen ratios of arterioles of three size classes (less than or equal to 15, 16-25, and 26-50 microns) were found to be nearly identical for the two age groups. Mean arteriolar diameter increased within the smallest size class in both ventricular regions. In contrast, a twofold or greater increase in wall thickness with age occurred in the left anterior descending coronary artery and its first branch, which was mainly due to expansion of the medial interstitium. The connective tissue fraction of the myocardium was significantly higher in the senescent than in the young in the epimyocardium and midmyocardium but not in the endomyocardium. These data provide evidence for three conclusions regarding aging of coronary vessels in beagles. First, a decline in capillary length density is limited to the endomyocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

The morphological structure of individual vascular smooth muscle cells from intestinal venules was evaluated with a combination of quantitative scanning (SEM) and transmission (TEM) electron microscopy techniques. In addition, growth of individual venular smooth muscle cells and of the overall vessel wall was compared from measurements of these variables during the rapid juvenile growth spurt from ages 4 to 6 and 10 to 12 weeks in Wistar-Kyoto rats. SEM revealed that smooth muscle cells of intestinal venules in weanling rats are very long (379 +/- 91 [SD] microns) and wide (6.0 +/- 1.3 microns) and very little further cell enlargement occurs during rapid juvenile growth. TEM studies indicated that passive inner vessel diameter and total muscle layer cross-sectional area of both the largest and intermediate diameter venules of young rats, as well as the percentage of the total wall area as muscle tissue in each venule type, did not significantly increase during body growth. These observations indicate that both the intestinal venules and their smooth muscle cells reach mature dimensions at a very early stage of life. Comparison of intestinal vascular smooth muscle cell dimensions indicates that venular smooth muscle cells are much larger in both cell length and volume than comparable arteriolar smooth muscle cells.

The purpose of this study was to evaluate microvascular development during normal skeletal muscle maturation and to determine the alterations associated with decreased blood flow caused by a decrease in demand. Unilateral orchidectomy was performed on 4-wk-old rats to reduce muscle tension and growth of one cremaster muscle. Three weeks later, total blood flow was reduced to 58 +/- 9% measured by the dual-slit velocity technique and 55 +/- 9% by radioactive microspheres, and the muscle was smaller when compared with the intact contralateral muscle. Blood flow per gram of tissue was not significantly different. Measured by closed-circuit television microscopy, the internal diameters and wall cross-sectional areas of all orders of arterioles (1A-4A), and the number of 4As per 3A had increased with age in the control muscle. The arcading arterioles increased in length by 35% as the intact muscle grew, but the number of 3As remained unchanged. Arteriolar length increased but not in proportion to muscle mass. As a result, large and small arteriolar density decreased with age. Thus, during normal skeletal muscle maturation, preexisting arterioles became elongated and only precapillary arterioles increased in number, resulting in a decreased ratio of arteriolar number to tissue mass. Unilateral orchidectomy inhibited the growth of arterioles in both size and number. A reduced diameter of the 1A in the orchidectomy muscle resulted in unchanged wall shear rate. Flow-induced shear stress and/or local changes in growth factors are suggested as possible mechanisms mediating the alterations.

Normotensive rats were chronically given 100 mg.kg-1.day-1 of captopril in their drinking water beginning 1 day before uninephrectomy. Mean blood pressure was significantly decreased 4 (17%) and 8 (18%) wk later in treated rats vs. age-matched controls. When histological techniques were used, a marked reduction of medial-intimal area of the abdominal aorta was found in treated rats at 4 (24%) and 8 (15%) wk without significant change in internal diameter. Cremaster arteriolar dimensions were measured by intravital microscopy after topical application of 10(-3) M adenosine. Structural diameter reductions occurred in large arterioles starting at 4 wk in treated rats and in small arterioles at 8 wk. Despite a significant increase in wall-to-lumen ratio of some large and small arterioles in treated rats, the wall cross-sectional area of these vessels was significantly decreased by captopril. Measured by stereological techniques, small arteriolar density decreased 17% at 4 wk and 13% at 8 wk in treated rats. Although decreased blood pressure may contribute to smaller aortic cross-sectional wall area, the effect of captopril was probably the major cause for smaller arteriolar lumens, reduced arteriolar cross-sectional wall areas, and arteriolar rarefaction.

The same region of intestinal ileum was compared in the same rats at 10 and 20 wk of age to determine if reported differences in vessel density between these ages were the result of changes in vessel numbers. The number and branching pattern of arterioles observed remained remarkably constant during the 10 wk between observations, and minimal enlargement of bowel mass occurred. No significant change in average maximum diameter of arterioles was observed. Although the total number of small arterioles did not change, vessels were gained and lost at an equal rate (7%) between 10 and 20 wk. There was no significant changes in means or frequency distributions of intercapillary distances in the radial or longitudinal bowel muscle layers. The results indicate that the intestinal microvascular branching pattern changes very little from juvenile to adult life. However, a slow turnover of small arterioles does occur, indicating an ongoing remodeling of the terminal vasculature during juvenile life.

This experiment was designed to investigate the effect of converting enzyme inhibition on functional and structural vascular alterations in one-kidney, one clip hypertensive rats and in normotensive rats. Starting 1 day before surgery, 100 mg/kg/day captopril was given chronically to half of the hypertensive and normotensive groups in their drinking water. With use of intravital microscopy in the cremaster muscle, arteriolar dimensions were measured 4 weeks later, both before and after topical application of 10(-3) M adenosine. Mean blood pressure was 124 +/- 4 mm Hg in control rats and 103 +/- 5 mm Hg in captopril-treated control rats (p less than 0.05). Mean blood pressure was significantly elevated to 183 +/- 5 mm Hg in captopril-treated one-kidney, one clip hypertensive rats and 193 +/- 5 mm Hg in one-kidney, one clip hypertensive rats. With use of histological techniques, a marked reduction of medial-intimal area of the abdominal aorta was found in captopril-treated control rats (24%), and hypertrophy of the aortic wall in one-kidney, one clip hypertensive rats was decreased 26% by captopril. Structural diameter reductions occurred in large arterioles of the captopril-treated control and hypertensive groups and the nontreated hypertensive group. In spite of a significant increase in wall-to-lumen ratio of first-order arterioles in all captopril-treated rats, captopril decreased cross-sectional wall area of these vessels 37% in hypertensive and 20% in control rats, respectively. Measured by stereological techniques, small arteriolar density decreased 30% in captopril-treated hypertensive rats and 17% in captopril-treated control rats. Therefore, smaller arteriolar lumens, decreased aortic and arteriolar cross-sectional wall area, and arteriolar rarefaction after converting enzyme inhibition, in spite of rising or falling blood pressure, are evidence that vascular growth was inhibited in vivo.

The process of revascularization of free grafts of the extensor digitorum longus muscle in rats has been studied by gross, histological and electron microscopic methods. During the first day after transplantation the muscle is entirely avascular, and it consists of a thin peripheral zone of surviving muscle fibers and a large central area of ischemic muscle. The original blood vessels of the graft undergo a sequence of intrinsic and cell-mediated destruction. Scattered sinusoidal vessels begin to grow into the graft starting on the second day, and ingrowing blood vessels progressively invade the deeper tissues of the graft. Most new vessels form in the connective tissues, but some vessels, especially larger ones, grow into persisting basal laminae from preexisting fibers and nerves. The differentiation of new arterioles and venules in free muscle grafts is described. By the end of the first week, the entire graft is revascularized, and ultimately a fairly normal relationship between new capillaries and regenerating muscle fibers is established. In mature grafts, however, irregularities are sometimes found in the organization of smooth muscle cells associated with larger vessels.

Long-term autoregulation of arterial blood flow to sustained increases or decreases in arterial pressure has been previously reported. It is not clear whether the adaptive process at the microscopic level is a modification of vessel caliber or density. The present study was undertaken to assess morphological and hemodynamic changes resulting from a sustained decrease in perfusion to the cremaster muscle in normal rats. The main feeding arteriole to the cremaster muscle was ligated at age 3 weeks in 11 male Wistar rats. At 6 weeks, the cremaster muscles on the ligated and control sides were evaluated microscopically for hemodynamic status and vascular architecture differences. Pressure was reduced 30% on the ligated side without a decrease in volume flow. This was possible because the number of second-order arterioles increased by 48% and the number of third-order arterioles increased by 98% on the ligated side. Although twice as many third-order arterioles were present in the ligated muscle, the same percentage was closed to flow in the resting state as in the control muscle (31 vs 29%). These findings are consistent with a long-term autoregulatory process which modulates arteriolar density in response to altered perfusion or distending pressure. The same process may be responsible for arteriolar rarefaction in response to sustained elevation in arterial pressure in the cremaster muscle of the spontaneously hypertensive rat (SHR).

The capillary network of the enamel organ of the continuously growing mandibular incisor of the rat is supplied by a series of arteries which, after penetrating the bone, are disposed in a row in the periodontium along the tooth and linked together by anastomoses. The branches from these arteries are subjected to adaptive changes consistent with a forward movement of the capillaries in relation to the arteries. The mechanism which renders this movement possible appears to be the following: Each artery in the row supplies a section of the passing plexus by coupling and uncoupling short-lived arterioles, which go through a cycle of proliferation, elongation and degeneration. Proliferation takes place at the posterior end of the section, where new arterioles replace discarded arterioles from the preceding artery. By growing in length, the arterioles keep pace with the migrating capillary network. When the capillaries are within reach of the next artery, the arterioles disconnect. Thereafter they become obliterated and die in the intermediate zone of the periodontium, to which they have been gradually displaced by proliferation of the inner-tooth-related layer of the periodontal connective tissue.

This study investigates the development of the vascular system of the puppy kidney (1-21 days after birth) after preparing casts of the renal vessels. At two days, the intrarenal vascular system distal to the afferent arteriole is strikingly different than that of the adult. The glomeruli of the outer cortex consist of a single dilated vessel while those of the mid and inner cortex posses an increasingly larger number of capillary loops. The efferent arterioles vary greatly in appearance from outer to inner cortex. Those in the nephrogenic zone are characteristically short and narrow and join a larger venous vessel termined a sinusoidal capillary. An efferent system somewhat similar to that of the adult is seen in the mid and inner cortex. One of the most obvious differences noted between the puppy and adult kidney is the relative lack of peritubular capillary networks throughout the cortex of the puppy kidney. The puppy possesses large, irregular vessels termed sinusoidal capillaries. The most rudimentary sinusoids are found in the outer cortex with more mature vessels in the inner cortex. The vascular arrangement of the efferent arteriole and sinusoidal capillary appears as a post-glomerular shunt. Functionally, the shunt would direct blood flow away from the proximal tubule and thus could result in a low extraction ratio and Tm for secreted solutes.

This study was undertaken to determine what changes occur in the intestinal microvasculature during the rapid growth associated with juvenile maturation. A technique was developed that permitted the comparison of the same microvessels in exactly the same intestinal region at two time periods of an animal's life. A region of the terminal ileum of 5-week-old rats was exposed and marked, and photographs and video recordings were made of the microvessels. Four weeks later, the marked intestinal region was located, and photography and videography of the microvessels were repeated. Comparison of indexes for body, intestinal, and microvascular growth for the treated rats and age- and colony-matched controls revealed no significant differences. The number and branching pattern of arterioles observed in the marked region remained remarkably constant during the 4 weeks between observation periods, even though body and bowel mass of the treated animals increased approximately 2.5 times. The lengths of the arterioles were increased (18%) by almost the same proportion as the axis of bowel (22%) in which they were oriented. The average distance between capillaries in the radial intestinal muscle layer was also increased by about the same percentage (24%) as that of tissue elongation (22%). The overall data are consistent with the hypothesis that during the growth spurt of juvenile life, the arterioles present at the weanling stage are elongated and new branches do not develop. The net effect of tissue growth with a minimal change in numbers of arterioles is a decreased ratio of number of arterioles to tissue mass as a normal consequence of maturation.

A study to determine the growth potential of immature microvessels was carried out using rat femoral arteries. Immature rat arteries repaired with interrupted 10-0 nylon sutures grew in proportion to the animal. There were no areas of stenosis at repair sites, which in some animals appeared slightly dilated compared with control vessels. The ultrastructure of repaired arteries after growth was mildly altered in the internal and external elastic membrane layers while the intima and media appear normal. Neovascularization around anastomoses may explain an apparent increased blood flow velocity at repair sites and does not correspond to arterial narrowing in this model.

The state of development of the intramyocardial coronary arterioles in normal hearts and hearts with congenital malformations is unknown, although these vessels play an important role in regulating myocardial blood flow. To remedy the situation, we studied 64 normal hearts (ranging in age from 23 weeks gestational age to 75 years), 13 hearts with complete transposition (5 with intact ventricular septum and 8 with a defect) and 11 hearts with classical aortic atresia. The medial thickness of arterioles with an external diameter from 20 to 100 microns was measured. The number of arterioles was calculated per mm2; a distinction was made between vessels between 20 and 100 microns and those smaller than 20 microns. The results show that the arteriolar vascular bed is not yet full grown at term. Within the first year of life the medial smooth muscle mass increases, most likely due to growth and peripheral extension onto the smaller branches. In the hearts of patients with aortic atresia who lived for a week or less, the arteriolar media appeared to lag behind that in normal hearts. In those patients who survived into the second post-natal week, however, the arteriolar bed was almost as anticipated for normal. The normal values obtained in the latter group could be due to intrinsic differences, rather than expressing actual and rapid growth.