Microvascular architecture in a mammary carcinoma: branching patterns and vessel dimensions.

The objective of this work was to introduce a tumor vessel classification scheme and to provide the first quantitative measurements of vessel branching patterns and the related vascular dimensions in a mammary carcinoma. Mammary adenocarcinoma R3230AC tumors, grown in the rat ovarian tissue-isolated tumor preparation, were infused with Batson's No. 17 polymer and maintained at an intravascular pressure of 50 mm Hg during polymerization. Maceration of the tumor in KOH allowed visualization of the vasculature. The vessel branching patterns, lengths, and diameters were measured in four tumors (4-5 g). A centrifugal ordering scheme was devised specifically to account for the unique features of tumor microvascular network topology. The arterial networks revealed two types of branching patterns. One type of arteriolar network exhibited decreasing vessel diameters and lengths with increasing branch order. In a second type of network, the diameter and length of the vessels displayed fluctuations in both variables at higher generations. Avascular and poorly vascularized regions with sparse capillary supply were present in the tumors, but analysis of several capillary networks in vascularized regions revealed a nonplanar meshwork of interconnected vessels. The meshworks were composed of vessels with a mean segment length of 67 microns, a mean diameter of 10 microns, and a mean intercapillary distance of 49 microns. Capillary path lengths ranged from 0.5 to 1.5 mm. Thus, tumor capillary diameter was greater than that in most normal tissues and, in the regions where capillary networks existed, intercapillary spacing was in the normal range. In the venous network, diameters decreased from 650 to 20 microns for the first to ninth order venules. Venule length decreased from 5 to 0.5 mm for first to fourth order but was fairly uniform (less than 500 microns) for higher orders. In conclusion, solid tumor vascular architecture, while exhibiting several features that are similar to those observed in normal tissues, has others that are not commonly seen in normal tissues. These features of the tumor microcirculation may lead to heterogeneous local hematocrits, oxygen tensions, and drug concentrations, thus reducing the efficacy of present day cancer therapies.

Hemodynamic characteristics, myocardial kinetics and microvascular rheology of FS-069, a second-generation echocardiographic contrast agent capable of producing myocardial opacification from a venous injection.

OBJECTIVES: We sought to 1) study the effects of FS-069 on cardiac and systemic hemodynamic function, myocardial blood flow, left ventricular wall thickening and pulmonary gas exchange when injected intravenously; and 2) compare the myocardial kinetics and microvascular rheology of FS-069 and Albunex when injected directly into a coronary artery. BACKGROUND: FS-069 is a second-generation echocardiographic contrast agent composed of perfluoropropane-filled albumin microspheres; it is capable of consistent and reproducible myocardial opacification from a venous injection. METHODS: Nine dogs were used to study the effects of FS-069 on hemodynamic function, pulmonary gas exchange, left ventricular wall thickening and myocardial blood flow and to characterize its myocardial kinetics when injected intravenously. These dogs were also used to compare the myocardial kinetics of FS-069 with those of Albunex during intracoronary injections. Nine Sprague-Dawley rats were used to compare the microvascular rheology of these two contrast agents, and in vitro modeling was performed to assess whether the microvascular findings of FS-069 can explain its echocardiographic behavior during direct coronary injections. RESULTS: There were no effects of 30 rapid venous injections of FS-069 (every 20 s) on cardiac output; mean aortic, pulmonary or left atrial pressures; and peak positive and negative first derivative of left ventricular pressure (dP/dt). Similarly, there were no effects of this agent on radiolabeled microsphere-measured regional myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange. When injected intravenously, the myocardial transit of this agent resembled a gamma-variate form. When diluted FS-069 was injected directly into the coronary artery; however, its transit resembled the integral of gamma-variate function, with persistent myocardial opacification lasting several minutes, which was different from that of Albunex. Intravital microscopy revealed that, unlike Albunex, when no bubbles are entrapped within the microcirculation after an arterial injection, a very small fraction of the diluted, larger FS-069 microbubbles are entrapped. In vitro modeling confirmed that this small fraction of microbubbles can result in persistent myocardial opacification. CONCLUSIONS: FS-069 produces no changes in hemodynamic function, myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange when injected intravenously in large amounts. When diluted FS-069 is injected into the coronary artery, a very small fraction of the larger bubbles are entrapped within the microcirculation, resulting in a persistent contrast effect. Thus, although FS-069 is a safe intravenous echocardiographic contrast agent, it cannot provide information on myocardial blood flow when injected directly into a coronary artery.

Spontaneous redistribution after reperfusion: a unique property of AIP 201, an ultrasound contrast agent.

OBJECTIVES: We sought to determine the mechanism of spontaneous redistribution of AIP 201 microbubbles after reperfusion from a single left heart injection performed during coronary occlusion. BACKGROUND: AIP 201, an ultrasound contrast agent consisting of 10-microm sized microbubbles, has demonstrated spontaneous myocardial redistribution in preliminary studies. METHODS: Myocardial video intensity (VI) and radiolabeled microsphere-derived myocardial blood flow (MBF) were measured serially after reperfusion in seven dogs undergoing an AIP 201 injection during coronary occlusion. The behavior of these bubbles was also assessed in the rat spinotrapezius muscle using intravital microscopy (IM), both with and without ultrasound. The effect of ultrasound on these bubbles was also determined in vitro. RESULTS: A spontaneous and gradual increase in myocardial VI was noted after reperfusion, which was related to the magnitude of increase in MBF to that region (r=0.82, p < 0.001). On IM, most of the microbubbles were seen entrapped in small arterioles. Some larger arterioles had aggregates of microbubbles that periodically became dislodged and moved downstream. This behavior was not affected in vivo by ultrasound. In vitro, however, microbubble aggregation was noted only during ultrasound exposure. CONCLUSIONS: The magnitude of redistribution of AIP 201 microbubbles to the reperfused myocardium is related to changes in MBF and occurs from their dislodgement from microbubble aggregates entrapped in large arterioles. In vitro microbubble aggregation seen during ultrasound exposure was not reproduced in vivo. These results may have important implications for studying the effects of interventions in acute coronary syndromes and after coronary artery bypass graft surgery.

Accuracy of the conductance catheter for measurement of ventricular volumes seen clinically: effects of electric field homogeneity and parallel conductance.

The conductance-volume method is an important clinical tool which allows the assessment of left ventricular function in vivo. However, the accuracy of this method is limited by the homogeneity of electric field the conductance catheter produces and the parallel conductance of surrounding structures. This paper examines these sources of error in volumes seen clinically. The characteristics of electric field within a chamber were examined using computer simulation. Nonconductive and conductive models were constructed and experimental measurements obtained using both single-field (SF) and dual-field (DF) excitation. Results from computer simulations and in vitro measurements were compared to validate the purposed theoretical model of conductance-volume method. The effects of field homogeneity and significance of parallel conductance in volume measurement were then determined. The results of this study show that DF provide a more accurate measure of intraventricular volume than SF, especially at larger volumes. However, both significantly underestimate true volume at larger volumes. In addition, the parallel conductance due to the chamber wall is significant at small volumes, but diminishes at larger volumes. Furthermore, the effect of parallel conductance beyond the chamber wall may be negligible. This study demonstrates the limitations in applying current conductance technology to patients with dilated hearts.

A dedication in memoriam of Dr. Richard Skalak.

Richard Skalak (1923-1997) played a leadership role in the formative decades of the discipline of biomedical engineering through his technical contributions in biomechanics, his educational influence on students, and his service to many developing societies and journals. But always, the distinguishing marks of his involvement with any activity or person were his generosity, respect and tolerance for others, integrity, and curiosity. These very qualities are what first brought him as a traditional engineering trained in engineering mechanics into the young field of biomedical engineering in the 1960s, and they are what led him to new approaches to cellular and molecular engineering, tissue engineering, and orthopedic biomechanics. His technical papers and lectures on blood cell mechanics, pulmonary circulation, dental implants, and tissue growth were models of clarity and often pointed the way to new areas of exploration, while his personal writings offer advice on life, academic organizations, and the pursuit of significant work. He would be deeply appreciative that this first volume of the Annual Review of Biomedical Engineering is dedicated to his memory.

Effects of leukocyte activation on capillary hemodynamics in skeletal muscle.

Leukocytes may be an important determinant of microvascular resistance, particularly during pathological states that cause cell activation and cytoplasmic stiffening. Significant mechanisms include capillary plugging and venular adhesion. Previous quantitative studies on leukocyte-capillary plugging focused solely on arteriolar-capillary branchpoints. There are no quantitative data on plugging throughout the capillary network either under normal conditions or after leukocyte activation. Plugging measurements were made throughout capillary networks at both arteriolar-capillary and capillary-capillary branchpoints in spinotrapezius muscle of anesthetized rats under normal physiological conditions and after leukocyte activation by superfusion with 1 x 10(-7) M N-formyl-methionyl-leucylphenylalanine (FMLP). These data were used to estimate the increase in microvascular flow resistance due to leukocyte plugging. The increase was 1.1% in control and 15.9% in the activated state. On an individual network basis, this represents an average 23-fold increase (P < 0.002) in network resistance, suggesting that leukocyte activation has a significant impact on microvascular blood flow.

A circumferential stress-growth rule predicts arcade arteriole formation in a network model.

OBJECTIVE: To test the hypothesis that terminal arteriolar remodeling that is stimulated by elevated levels of circumferential wall stress (sigma theta) will proceed in a network pattern that gives rise to new arcade arterioles. METHODS: A network model of two interconnected skeletal muscle arterio-capillary-venous units that incorporated diameter- and hematocrit-dependent blood viscosity was constructed. After computing the control values for wall shear stresses (tau ij) and sigma theta ij, a stimulus was provided by dilating the arterioles and raising input pressure. Wall shear stresses and sigma theta ij were then recomputed. The diameters of transverse arteriolar segments with sigma theta ij greater than a sigma theta threshold were increased by an amount that was dependent on the original diameter and the difference between sigma theta ij and the sigma theta threshold. Capillaries with an intraluminal pressure greater than a specified threshold were converted to terminal arterioles. Separate simulations in which remodeling was stimulated by elevated levels of tau ij were also performed for comparison. RESULTS: Arterialization patterns from simulations of sigma theta ij stimulated arteriolar remodeling were representative of those seen in vivo with arterialization of back-connection capillaries leading to arcade arteriole formation. Simulations based on similar rules for tau ij yielded arterio-venous shunts, which are rarely seen in vivo, but no arcade arterioles. CONCLUSION: The simulations presented here are consistent with the hypothesis that arteriolar remodeling is stimulated by increased levels of circumferential wall stress and that new arcade arteriole formation is a consequence of terminal arteriolar growth.

Circumferential wall stress as a mechanism for arteriolar rarefaction and proliferation in a network model.

Hypertension results in structural rarefaction of the microvascular arteriolar network while, conversely, decreasing pressure results in arteriolar proliferation. A remodeling mechanism capable of unifying these results remains elusive. A network model of a transverse arteriole tree was used to test whether adaptations to changes in mean circumferential wall stress (sigma theta) could produce realistic structural remodeling. Vessel diameters and network boundary pressures were assigned using experimental data, and control flows (Qij) and sigma theta ij for each vessel were calculated. Mean sigma theta ij in A2 (Strahler order) vessels (sigma m) was 6.62 x 10(4) dynes/cm2. Input pressure was increased by 35% in simulated one-kidney, one-clip (s1K1C) hypertension or decreased by 30% in simulated main feeder ligation (sMFL). Vessel diameters were adjusted iteratively until each Qij was restored, simulating autoregulation. Wall stresses decreased 15.9% for hypertension (sigma m = 5.57 x 10(4) dynes/cm2), but were elevated 60.9% for main feeder ligation (sigma m = 10.65 x 10(4) dynes/cm2), A stress-growth principle was applied, so that stresses above an upper threshold cause growth while stresses below a lower threshold cause resorption. Individual vessels with sigma theta ij < 5.24 x 10(4) dynes/cm2 were removed while new A2 segments were added to A2 vessels with sigma theta ij > 8.27 x 10(4) dynes/cm2. The network structure was adjusted until all sigma theta ij were within these stress thresholds. The number of A2s decreased 22% in s1K1C and increased 96% in sMFL in quantitative agreement with experimental data, consistent with the hypothesis that wall stress may be an important determinant of network remodeling. Arteriolar proliferation and rarefaction represent adaptations to different hemodynamic conditions, but may be governed by a common stress-growth principle.

Leukocyte cytoskeletal structure determines capillary plugging and network resistance.

Leukocyte plugging in capillaries may play an important role in microvascular hemodynamics under pathological conditions that result in leukocyte activation. Previous studies have quantified the effects of transient capillary plugging on microvascular resistance for control and activated leukocytes, but have not focused on the intracellular mechanisms responsible. The frequency and duration of leukocyte-capillary plugging were measured throughout capillary networks in spinotrapezius muscle of anesthetized rats under normal physiological conditions and after leukocyte activation coupled with cytoskeletal F-actin depolymerization, using superfusion with 10(-7) M N-formyl-methionyl-leucyl-phenylalanine (FMLP) and 0.3 microM cytochalasin D (CD). These data were used to estimate the increase in microvascular flow resistance due to leukocyte plugging. The increase was 1.1% in control and 0.7% in the activated FMLP-CD state and was not significantly different. CD abolished the previously reported 16% resistance increase caused by FMLP activation [Harris and Skalak, Am. J. Physiol. 264 (Heart Circ. Physiol. 31): H909-H916, 1993.], suggesting that leukocyte plugging in capillaries is primarily determined by leukocyte cytoplasmic viscosity and is not significantly affected by cell adhesion. Thus F-actin-mediated cytoskeletal reorganization during leukocyte activation has significant impact on microvascular resistance.

In vivo measurement of leukocyte viscosity during capillary plugging.

Leukocyte plugging of capillaries in vivo was measured in rat spinotrapezius muscle. The plug durations, leukocyte and capillary dimensions, and arteriolar pressure at the plug sites were applied to the mechanical model of Needham and Hochmuth (1990) to estimate the leukocyte viscosities. The viscosity distribution of 389 cells was lognormal with a median value of 232 Poise. 3.1 percent of the cells were apparently activated and displayed viscosities greater than 3000 Poise. The median viscosity suggests that inactivated leukocytes have a minimal effect on blood flow, but that leukocyte activation may result in significant increases in microvascular flow resistance.

Leukocyte plugging in vivo in skeletal muscle arteriolar trees.

To provide quantitative data on leukocyte plugging and to estimate its effects on blood flow resistance in the microcirculation, in vivo observations of leukocyte plugging of capillaries were made in all branches of 27 terminal arteriolar trees in spinotrapezius muscles of anesthetized Sprague-Dawley rats. The durations of 1,257 observed plugs had a lognormal distribution with a median of 0.12 s. Of 596 branches, 211 experienced at least one plug; this subpopulation had a median plugging frequency of 0.016/s and a median plugging fraction (plugging frequency times average duration) of 0.28%. A new variable, the network occlusion fraction, was defined to quantify plugging in a whole arteriolar tree. Applying in vivo data to a model relating plugging to resistance increases resulted in a lognormal distribution of resistance increases with a median of 1.6%. Each of the resistance increases that were greater than 3% was due to one or two individual leukocytes that plugged a capillary for greater than 30 s. The results suggest that, under physiological conditions, leukocyte plugging has little effect on blood flow resistance.

The role of mechanical stresses in microvascular remodeling.

The microvasculature is an extremely adaptable structure that is capable of architectural and functional adjustments in response to multiple biochemical and mechanical stimuli. Inadequate or inappropriate adjustments often result in pathophysiology. Recent work has brought increasing recognition of the importance of microvascular remodeling in widespread disease states such as hypertension, tumor growth, diabetes, and progressive coronary artery occlusion. Much work has been done to characterize the cells and molecules with putative roles in microvascular remodeling, but little is known regarding the mechanotransduction processes that might link hemodynamic stresses such as wall shear stress and circumferential wall stress to structural and functional changes in vivo. Two primary approaches have been employed: in vitro studies that use cultured cells and allow molecular biologic analysis of signaling pathways and gene expression; and in vivo experiments aimed at understanding vessel adaptations in the intact tissue. This article reviews the structural adaptations exhibited by microvessels and the information available from in vitro and in vivo approaches. The formation of new arterioles in intact tissues is examined in detail as an example of integrative work, and the prospects for new technologies are discussed. This is a time of great opportunity for bidirectional exchange between basic in vitro advances and in vivo experimentation. This exchange will be essential in generating new understanding of the role of mechanical stresses in microvascular remodeling.

Chronic alpha 1-adrenergic blockade stimulates terminal and arcade arteriolar development.

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.

Effects of leukocyte capillary plugging in skeletal muscle ischemia-reperfusion injury.

The purpose of this study was to examine the relationship between increased capillary network resistance due to leukocyte capillary plugging and tissue injury following ischemia-reperfusion (I/R). After a 30-min complete ischemia in rat spinotrapezius muscle, the frequency and duration of leukocyte capillary plugging were measured throughout capillary networks and used to estimate the increase in network flow resistance for I/R alone, I/R with phalloidin (Pl), and I/R with both Pl and cytochalasin D. Propidium iodide (PI) was used to label nonviable muscle cell nuclei within the volume of tissue supplied by the capillary network, and counts were made before ischemia, immediately after reperfusion, and 1 h postreperfusion. For I/R alone and I/R + Pl there is a linear correlation between the increase in resistance (up to 29%) and the increase in the number of PI-positive nuclei during the reperfusion period. With both Pl and cytochalasin D present in the superfusate, the resistance increase was abolished and the amount of tissue damage during reperfusion was minimized. The results indicate that the increase in resistance is linearly related to the tissue damage and that a reduction of the leukocyte stiffness reduces the injury.

Role of leukocytes and tissue-derived oxidants in short-term skeletal muscle ischemia-reperfusion injury.

The relative contribution of xanthine oxidase (XO) and leukocytes to tissue injury after short-term ischemia is unknown. In this study, we subjected three groups of rat spinotrapezius muscles to 30-min ischemia and 1-h reperfusion: 1) ischemia-reperfusion (I/R) + 0.9% saline, 2) I/R + superoxide dismutase, and 3) I/R + oxypurinol. A fourth group served as nonischemic control. We quantified the increase in resistance (%DeltaR) caused by leukocyte-capillary plugging concurrently with myocyte uptake of propidium iodide (PI) [expressed as no. of PI spots per total volume of perfused tissue (N(PI)/V)] and performed assays to quantify XO activity, thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase (MPO). Groups 2 and 3 exhibited significant decreases in N(PI)/V relative to group 1. MPO levels and TBARS were similar among all groups, and mean %DeltaR was significantly reduced in groups 2 and 3 relative to group 1. However, elevated XO was observed in groups 1 and 2 relative to group 3 and nonischemic controls. These data are consistent with the hypothesis that XO, rather than toxic species produced by plugging or venule-adherent leukocytes, is responsible for postischemic damage in this model.

Contribution of individual structural components in determining the zero-stress state in small arteries.

Arterial-wall stress distributions are important determinants of arterial function and are determined by the mechanical properties of the vessel wall, the physiological loading conditions, and the zero stress state of the artery. In this study, the relationship between this zero-stress state, the contractile state of the vessel, and the extracellular matrix components was investigated. Rat saphenous arteries were excised, and cross-sectional slices were cut. Wall thickness, intimal circumference, medial to adventitial border circumference, and the outer adventitial circumference were measured. Each slice was cut once longitudinally to relieve the residual stresses, and after a period of 30 min, vessel dimensions were again measured, and the angle to which the arterial section opened, a measure of the residual stress present in the intact artery, was recorded in this new zero-stress state. Control 'opening angle' was 112+/-10 degrees (mean+/-SD, n= 8) as compared to 138+/-6 degrees (n = 18) and 127+/-6 degrees (n = 11) for slices placed in 10(-4) M adenosine at 25 and 37 degrees C, respectively. Fluorescein isothiocyanate dextran was also injected intravenously in 8 animals to measure the homeostatic lumen diameter and wall thickness using intravital microscopy. Comparison of homeostatic strains calculated from these in situ dimensions to residual strains indicated that a given opening angle produced a radially uniform circumferential strain only for a specific level of tone. These results suggest that smooth muscle tone can acutely modify residual strain, possibly through interconnections with matrix components. Furthermore, selective elimination of matrix components by post-treatment of zero-stress slices with elastase or collagenase in 6 animals each increased the opening angle by 53 and 70%, respectively, suggesting that the extracellular matrix structure may regulate arterial strains in the long term.

Arteriolar remodeling in skeletal muscle of rats exposed to chronic hypoxia.

The topological structure of the arteriolar network is an important determinant of microvascular resistance. In several normal and pathological conditions, this network topology is remodeled such that adequate tissue perfusion and oxygenation are maintained. The objective of this study was to quantify the extent of arteriolar remodeling in skeletal muscle of rats chronically exposed to 10% oxygen for 18 +/- 3.6 days. Arcade arteriolar (AA) and transverse arteriolar networks that had been immunofluorescently labeled for smooth muscle alpha-actin and smooth muscle myosin heavy chain were observed in whole-mount gracilis muscles from hypoxic and weight-matched control rats. Eight muscles from 4 animals were used for analysis in each group. The percentage (+/- SD) of terminal arteriolar endings that were positive for smooth muscle alpha-actin, but negative for smooth muscle myosin heavy chain increased significantly (p < 0.05) from 29.2 +/- 8.0 in controls to 70.7 +/- 9.0 in hypoxia, indicating that the rate of terminal arteriolar development was elevated in the hypoxic animals. The number of AA loops/muscle (+/- SD) increased significantly from 9.6 +/- 2.3 in controls to 14.4 +/- 4.6 in hypoxia. This increase in AA loops/muscle was due primarily to a large increase in the number of small-diameter (<15 microm) AA segments/muscle, suggesting that new AA loops were formed via the recent anastomosing of developing small-diameter terminal arterioles. The results demonstrate that exposure to chronic hypoxia stimulates significant remodeling of both the arcade and transverse arteriolar networks in skeletal muscle.

Distribution of cellular proliferation in skeletal muscle transverse arterioles during maturation.

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.

Prazosin administration enhances proliferation of arteriolar adventitial fibroblasts.

Chronic vasodilation stimulates the formation of new arterioles in skeletal muscle, a process that requires the differentiation of mesenchymally derived precursor cells on the abluminal surface of capillaries. Fibroblast proliferation and migration to the arterializing capillary likely precede this differentiation process. In the current study, we investigated the effects of chronic vasodilation with the alpha1 adrenergic blocker prazosin, a treatment that produces enhanced terminal arteriolar development, on the proliferation of fibroblasts present in the adventitia of transverse arterioles. Dual-immunofluorescence labeling for the smooth muscle contractile protein SM-myosin heavy chain (MHC) and for bromodeoxyuridine (BRDU) uptake revealed that prazosin treatment for 4 days stimulated a threefold increase in the density of proliferating fibroblasts surrounding transverse arteriolar trees. This increase was primarily due to an eightfold increase in the density of S-phase fibroblasts surrounding <8 micron m diameter terminal arterioles and a 280% increase in the density of S-phase fibroblasts surrounding 8- to 12-micron m terminal arterioles. Alcian blue counterstaining indicated that no proliferating cells were mast cells. An in vitro study demonstrated that prazosin, at concentrations of 0.5 and 0.05 mg/liter, has no direct effect on fibroblast proliferation. It is concluded that chronic vasodilation with prazosin, a treatment that elicits elevated levels of hemodynamic stress, stimulates the proliferation of adventitial fibroblasts, particularly at the terminal endings of transverse arteriolar trees.