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Prazosin Administration Enhances Proliferation of Arteriolar Adventitial Fibroblasts
Microvascular Research 55, 138 – 145 (1998) Article No. MR972062
Prazosin Administration Enhances Proliferation of Arteriolar Adventitial Fibroblasts Richard J. Price and Thomas C. Skalak Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia Received October 17, 1997
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 a1 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 õ8 mm diameter terminal arterioles and a 280% increase in the density of S-phase fibroblasts surrounding 8- to 12-mm 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. q 1998 Academic Press
Key Words: microcirculation; arteriolar development; angiogenesis; hemodynamic stress.
INTRODUCTION
New arterioles form in skeletal muscle when mesenchymal precursor cells on the abluminal surface of capillaries differentiate toward a smooth muscle phenotype (Stingl and Rhodin, 1994; Price et al., 1994). In previous studies, it was determined that both the rate of terminal arteriolar development (Price et al., 1994) and the density of proliferating cells surrounding terminal arteriole endings is greater in 4week-old weanling animals when compared to 9week-old juvenile animals (Price and Skalak, 1998). By eliminating the possibility that the proliferating adventitial cells were mast cells, it was concluded that these cells were most likely fibroblasts. Because rates of arteriolar development and fibroblast proliferation are correlated during maturation, these results are consistent with a role for fibroblast proliferation in the assembly of new terminal arterioles. We hypothesize that fibroblast proliferation, and perhaps migration to the arterializing capillary, precede the differentiation of these precursor cells into smooth muscle. Several previous studies indicated that elevated levels of hemodynamic stress in individual capillaries are capable of stimulating their transformation into new 0026-2862/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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Vasodilation Enhances Fibroblast Proliferation
terminal arterioles (Price and Skalak, 1994, 1996; Skalak and Price, 1996). For instance, terminal arteriolar development was enhanced and collateral vessel formation was increased in animals that were chronically vasodilated with the a1 adrenergic blocker prazosin (Price and Skalak, 1996). A similar result was obtained by Dusseau and Hutchins (1979) who observed an increase in the number of skeletal muscle terminal arterioles in animals that had received the b2 agonist salbutamol, a vasodilator. The objective of the current study was to test the hypothesis that chronic vasodilation with prazosin elicits enhanced proliferation of interstitial cells surrounding transverse arterioles. The density of S-phase cells surrounding small diameter terminal arterioles was significantly increased in animals treated with prazosin for 4 days, indicating that elevated levels of hemodynamic stress stimulate the proliferation of interstitial fibroblasts which may subsequently invest arterializing capillaries.
MATERIALS AND METHODS
(2) washing briefly in buffer, (3) incubating for 1 h at 377C in a 0.2 mg/ml solution of pepsin in 0.1 N HCL, and (4) incubating in 1 N HCL for 30 min at 377C. For two of the prazosin specimens, 1% Alcian blue was added to the incubating solution in step 3 above. Before starting the immunochemistry protocol, specimens were washed thoroughly in buffer. A stock solution of 0.4% (vol/vol) Triton X-100, 3% bovine serum albumin, and 5% (vol/vol) normal goat serum was used in each antibody incubation. Incubations lasted for 24 h and were done at room temperature. Concentrations are given in each step. Incubations proceeded in the following order: (1) Mouse monoclonal Ab to bromodeoxyuridine (1:40), (2) Goat anti-mouse IgG Fab fragments conjugated to fluorescein isothiocyanate (FITC, 1:100), (3) Hybridoma supernatant containing mouse monoclonal Ab to SM-MHC (1:1), and (4) Goat anti-mouse IgG Fab fragments conjugated to lissamine rhodamine sulfonyl chloride (LRSC, 1:100). The mouse monoclonal Ab to BRDU was from Dako Inc., the mouse monoclonal Ab to SM-MHC was developed at the University of Virginia hybridoma facility by Dr. Gary K. Owens, and the secondary Abs (Fab fragments) were from Jackson Immunoresearch Laboratories Inc.
Specimen Preparation Female Sprague – Dawley rats (n Å 5) received prazosin (50 mg/liter) in their drinking water for a period of 4 days. Treated and weight matched control animals (n Å 5) were anesthetized with an intramuscular injection of 13.3% urethane and 1% a-chloralose (0.6 ml/ 100 g). The right gracilis muscle was exposed by removing the overlying skin and fascia. Regions surrounding the gracilis muscle were packed in cotton to retain moisture. Bromodeoxyuridine (1005 M in Ringer’s solution) was administered to the muscle by superfusion at a flow rate of 4 ml/min. After 2 h of superfusion with the Ringer’s solution containing BRDU, a descending catheter was placed in the right saphenous artery. To fix the gracilis muscle in its in situ position, a solution of 3% paraformaldehyde in phosphate buffer was perfused through the saphenous artery catheter for 5 min. The muscle was then washed by perfusion with phosphate buffer. Muscles were dissected free and processed for immunochemistry by: (1) incubating in a 3 mg/ml solution of collagenase in buffer for 30 min at room temperature,
Measurements and Cell Identification After identifying TA trees with an LRSC filter setting and a low power (120) objective, a dual LRSC-FITC filter setting and a 163 objective were used to measure the length and outer diameter of each clearly visible TA segment and to identify S-phase adventitial cells. S-phase adventitial cells were defined as those cells that were both negative for SM-MHC and contained a BRDU-positive nucleus within 5 mm of the arteriolar outer wall. To determine if any S-phase adventitial cells were mast cells, 2 of the 5 prazosin specimens were counterstained with Alcian blue, which is capable of labeling mast cells in S-phase (Osinski et al., 1993). Sphase mast cells were, therefore, designated as those mast cells with an intracellular region of positive FITC labeling within 5 mm of the arteriolar outer wall. Proliferating smooth muscle and arteriolar endothelial cells were not included in this study because, consistent with an earlier study on cell proliferation during maturation (Price and Skalak, 1998), only a few were observed in Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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each group, and no meaningful conclusions could be made by counting these cell types. S-phase adventitial cell densities were calculated as the number of S-phase cells per unit surface area, as determined by the dimensions of the individual TA segments. S-phase cell densities were further reduced into two forms. In the first form, the density of S-phase cells per TA tree was calculated such that the n value was the total number of TA trees in each group. This data was compiled in a histogram format, and the mean value for the prazosin group was compared to the mean of the control group using a student’s t test at the P õ 0.05 level. In the second form, S-phase cell densities were determined for various diameter ranges throughout the TA trees. The following diameter ranges were chosen; õ8 mm, 8.1 – 12 mm, 12.1 – 16 mm, 16.1 – 20 mm, 20.1 – 24 mm, 24.1 – 28 mm, and ú28 mm. Here, n values were the total number of observed TA segments for each group. The means from each diameter range in the prazosin group were then compared to control for the same diameter range using an unpaired student’s t test with significance assessed at the P õ 0.05 level. For the Alcian blue counter stained specimens, the percentage of S-phase interstitial cells that were also mast cells was determined.
In Vitro Fibroblast Proliferation Assay To study the direct effects of prazosin on fibroblast DNA synthesis, fibroblasts were incubated in media containing prazosin and subsequently assayed for BRDU uptake. The in vitro protocol was designed to match, as closely as possible, the in vivo experimental conditions and immunoassay procedures. Rat fibroblasts were obtained by incubating skin explants in Dulbecco’s Modified Eagle Media containing 10% Fetal Bovine Serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 250 ng/ml amphotericin B for approximately 1 week. This period of time was sufficient for fibroblasts to migrate from the skin explant to the culture dish and become confluent in the region surrounding the explant. After removing the skin explants from the dish, fibroblasts were removed by trypsinization. Cultured fibroblasts were passaged three times in the media described above before being seeded into 35mm-diameter culture dishes at a concentration of 10,000 cells per dish. In preliminary experiments, this seeding
density was determined to yield approximately 80% confluency after 4 days. Because cell proliferation is inhibited when cells become confluent, a low seeding density was required to keep cells subconfluent during these studies. Experimental groups were: (1) media containing 0.5 mg/liter prazosin (n Å 10), (2) media containing 0.05 mg/liter prazosin (n Å 10), and (3) control media (n Å 10). Prazosin was dissolved in dimethylsulfoxyide (DMSO) before being added to culture media. Because the final vol/vol concentration of DMSO in prazosin treated media was 0.01%, 0.01% DMSO was added to control media as well. After 4 days, 1005 M BRDU was added to the culture media for 2 h. Media was then removed from the culture dishes and cells were washed in phosphate buffer before they were fixed in 3% paraformaldehyde for 5 min. A brief buffer wash was followed by incubations in 0.1 N HCl for 1 h and 1.0 N HCl for 30 min. Preliminary studies revealed that pepsin digestion was not required for the in vitro labeling. Antibody incubations were performed in the same stock solution as used in the in vivo assay. The first incubation was in the mouse monoclonal Ab to BRDU (1:100) for 2 h at room temperature. Following a wash in phosphate buffer, cells were then incubated for 1 h in goat anti-mouse IgG Fab fragments conjugated to FITC (1:200). For illustrative purposes, one dish from each group was also labeled for the intermediate filament vimentin using an antibody (clone V9 at a concentration of 1:100) that was directly conjugated to CY3. The vimentin Ab was from Sigma, Inc. To quantify fibroblast DNA synthesis, the total number of BRDU-positive nuclei per unit surface area was determined for each dish. Twenty randomly selected fields of view per dish were chosen for observation using an FITC filter and a 110 objective. After videotaping each field of view, the images were digitized and analyzed using the Optimas (v 6.1) software package. The total number of BRDU-positive nuclei per field of view was counted using the Optimas software and then scaled so that the reported result was the number of BRDU-positive nuclei per unit surface area. The mean number of BRDU-positive nuclei per dish was calculated for each group.
RESULTS On the day of the experiments, prazosin-treated and weight-matched control rats weighed ({SD) 216.8 {
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FIG. 1. Photomicrographic montage of a region of rat gracilis muscle from a prazosin-treated animal. (A) Transverse arteriole segment immunolabeled for SM-MHC with LRSC. (B) S-phase adventitial nucleus immunolabeled for BRDU with FITC. (C) Mast cells visualized under transmitted light with Alcian blue counterstaining. (D) Composite image of parts A, B, and C. Bar indicates 20 mm.
8.1 and 206.4 { 10.2 g, respectively. An example of the immunolabeling used in the in vivo studies is provided in Fig. 1. In Fig. 1A, a transverse arteriole that has been labeled for SM-MHC with a secondary Ab conjugated to LRSC is shown. The smooth muscle wrapping pattern is clearly evident and smooth muscle nuclei are visible as oblong or circular gaps in the SM-MHC labeling. Figure 1B depicts an S-phase cell that has been labeled for BRDU using antibodies conjugated to FITC. The punctate chromatin pattern is evident in this cell nucleus, which lies within 5 mm of the TA segment shown in Fig. 1A. Although S-phase interstitial cell nuclei were often oblong and parallel to the longitudinal axis of the nearby arteriole, such as in Fig. 1B, several shapes and orientations were observed. This particular specimen was also counterstained for mast cells using
Alcian blue. Three mast cells, shown in Fig. 1C, are evident in this region when viewed under transmitted light. In Figure 1D, the images from A, B, and C have been combined to illustrate the relative positions of the mast cells, S-phase nucleus, and TA segment. Note that the S-phase nucleus was not a mast cell nucleus. In fact, of the 125 nuclei within 5 mm of TA segments observed in the two Alcian blue counterstained specimens, none were mast cell nuclei. A few S-phase mast cells were, however, observed in other regions of the muscle. Figure 2 depicts the S-phase interstitial cell density data when defined as the number of S-phase interstitial cells per unit surface area of TA (cells/mm2). The mean density { SE was 34.97 { 3.04 (n Å 165 trees) for the prazosin group and 11.06 { 1.43 (n Å 160 trees) for the control group. These values are statistically different at Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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BRDU-positive nuclei per unit surface area for each of the three groups. No significant differences were found between groups, indicating that prazosin treatment for 4 days had no effect on fibroblast DNA synthesis.
DISCUSSION
FIG. 2. S-phase adventitial cell density histogram. Transverse arteriolar trees completely lacking S-phase adventitial cells are represented as the first bar along the horizontal axis (i.e., at the ‘‘0’’ tick). *Mean S-phase cell density per TA tree was significantly greater at the P õ 0.05 level for the prazosin group (n Å 165 TA trees) when compared to control (n Å 160 TA trees).
P õ 0.05. Figure 3 depicts S-phase interstitial cell densities throughout a range of TA outer diameters for both groups. For the control and prazosin groups, respectively, the õ8-mm range consists of 311 and 442 segments, the 8- to 12-mm range consists of 710 and 754 segments, the 12- to 16-mm range consists of 535 and 540 segments, the 16- to 20-mm range consists of 272 and 284 segments, the 20- to 24-mm range consists of 126 and 166 segments, the 24- to 28-mm range consists of 28 and 58 segments, and the ú28-mm range consists of 16 and 49 segments. The prazosin group exhibited a statistically significant eightfold increase from control in the density of S-phase cells surrounding TA segments less than 8 mm in diameter. In the 8- to 12-mm diameter range, a statistically significant 289% increase above control was observed for the prazosin group. No other significant differences were observed. Figure 4 illustrates the general appearance of the in vitro rat fibroblast cultures following immunolabeling for nuclear BRDU uptake using Abs conjugated to FITC. In Figs. 4A and 4B, respectively, S-phase nuclei are apparent in control cultured cells and cultured cells exposed to 0.5 mg/liter prazosin for 4 days. Immunolabeling for the intermediate filament vimentin using a CY3 conjugated Ab is shown in Figs. 4C and 4D for control and prazosin-treated cells, respectively, to illustrate that the fibroblasts were subconfluent after the 4day time period. Figure 5 depicts the mean number of
The objective of this study was to determine whether chronic vasodilation with the a1 adrenergic blocker prazosin enhances the proliferation of adventitial cells surrounding transverse arterioles. The density of S-phase interstitial cells within 5 mm of transverse arterioles was calculated using measurements taken from specimens that were dual-immunolabeled for SM-MHC and BRDU. Results indicated that the overall density of Sphase adventitial cells was increased in prazosintreated animals. This increase was primarily due to an increased density of cells surrounding small diameter (õ12 mm) terminal arterioles. Alcian blue counterstaining revealed that the proliferating cells were not mast cells, indicating by process of elimination that these cells were most likely fibroblasts. In vitro experiments demonstrated that prazosin treatment had no direct effect on fibroblast proliferation. Because prazosin treatment increases microvascular pressure and
FIG. 3. Bar graph of S-phase adventitial cell densities for various diameter ranges. Number of transverse arteriole segment observations (n) for control and prazosin groups, respectively, in each diameter range: õ8 mm, 311 and 442; 8 – 12 mm, 710 and 754; 12 – 16 mm, 535 and 540; 16 – 20 mm, 272 and 284; 20 – 24 mm, 126 and 166; 24 – 28 mm, 28 and 58; ú28 mm, 16 and 49. *Significantly different than control (same diameter range) at P õ 0.05.
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FIG. 4. Photomicrographs of immunofluorescently labeled rat dermal fibroblasts. (A and B) Control cultured fibroblasts (A) and fibroblasts exposed to 0.5 mg/liter prazosin for 4 days (B) labeled for BRDU uptake using a FITC conjugated secondary Ab. (C and D) Same regions of control and prazosin-treated cells as shown in (A and B), respectively, labeled for the intermediate filament vimentin using a primary Ab conjugated to CY3. After 4 days, cells were approximately 80% confluent. Bar indicates 30 mm.
flow, these results are consistent with the hypothesis that increased hemodynamic stress stimulates the proliferation of adventitial fibroblasts which may subsequently differentiate into smooth muscle. The current study was primarily undertaken to link the results of two previous studies performed in our laboratory. In the first study (Price and Skalak, 1996), prazosin treatment for 1 week stimulated the formation of new terminal arterioles in skeletal muscle. After 2
weeks of treatment, terminal arteriole development remained increased and a significant increase in the number of small diameter (õ15 mm) arcade arterioles was also observed. Similar results were obtained by Dusseau and Hutchins (1979) who observed that chronic administration of salbutamol, a b2 agonist and vasodilator, caused a 22% increase in the total number of small arterioles in Wistar – Kyoto rat cremaster muscle. In the second study (Price and Skalak, 1998), it was deterCopyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 5. Bar graph of in vitro S-phase cell densities for control fibroblasts, fibroblasts exposed to 0.5 mg/liter prazosin for 4 days, and fibroblasts exposed to 0.05 mg/liter prazosin for 4 days. No significant differences were observed between groups. n Å 10 dishes for each group.
mined that a relatively higher density of S-phase fibroblasts exists in the region surrounding terminal arterioles of 4-week-old weanling animals when compared to 9-week-old juvenile animals. Because the later result indicates that the density of S-phase adventitial fibroblasts correlates with the rate of arteriolar development during maturation, we hypothesized that fibroblast proliferation may be a component of capillary arterialization. Thus, the current study was designed to determine whether the correlation between increased Sphase adventitial fibroblast density and terminal arteriolar development is also present when prazosin is used as a stimulus for arteriolar growth. Indeed, the results clearly indicate that a population of transverse arteriolar adventitial cells, which are likely fibroblasts, proliferate in response to this treatment. Moreover, the results from this study are consistent with the previous study of S-phase adventitial cell proliferation (Price and Skalak, 1998) because enhanced proliferation of adventitial cells was greatest in the smallest diameter terminal arterioles. The local stimulus for adventitial cell proliferation generated by the prazosin treatment was most likely an increase in either wall shear stress or circumferential wall stress in the capillaries of treated rats. Dawson and Hudlicka (1989) and Ziada et al. (1989) have previously shown that capillary angiogenesis is stimulated in muscles that experience enhanced capillary blood flow in response to chronic treatment with pra-
zosin. Microvascular pressure is also elevated in capillaries and terminal arterioles of treated rats because, in a previous study (Price and Skalak, 1996), no change in systemic pressure was observed. The lack of a systemic pressure drop, in conjunction with vasodilation, indicates that the microvascular pressure gradient was shifted such that capillaries and terminal arterioles experienced greater pressures and circumferential wall stresses. Increased hemodynamic stresses (shear stress and stretch) are capable of triggering the production of mitogenic growth factors by the endothelium (Hsieh et al., 1992; Skalak and Price, 1996) and by smooth muscle (Wilson et al., 1993; Skalak and Price, 1996). In addition, Pearce and Hudlicka (1996) have observed an increase in the density of proliferating interstitial cells in skeletal muscle in response to electrical stimulation, an intervention that produces increased capillary wall shear stress and circumferential wall stress. Alterations in nitric oxide (NO) levels associated with changes in hemodynamics may also influence cell proliferation in this study. The potential effects of NO could be tested in the future using a NOS inhibitor. Although prazosin was chosen as the vasodilator for this particular study, alternative vasodilators such as nitrates, yohimbine, or hydralazine are good candidates for producing similar levels of hemodynamic stress and, potentially, arteriolar remodeling. While increased levels of hemodynamic stress appears to be the most likely explanation for the current results, there are potential confounding factors involved in this experiment. These factors include the effect of altered blood pressure on circulating levels of humoral factors, the effect of vasodilation on tissue oxygenation, and the direct effect of prazosin treatment on fibroblast proliferation. First, the effect of prazosin treatment on circulating ANG II, a mediator of microvascular growth and rarefaction (Hernandez et al., 1992), is unclear in this study because an inhibitor of ANGII, such as captopril, was not used. We have provided a detailed discussion of the potential role of ANG II in the prazosin treatment in an earlier study (Price and Skalak, 1996). Briefly, because decreased blood pressure is a stimulus for ANG II production and blood pressure throughout the experiment was unknown, the potential for ANG II production at various times during treatment clearly exists. However, in the earlier study (Price and Skalak, 1996), no significant decrease in
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blood pressure was observed after 7 days, indicating that when this particular dosage is provided for short periods of time, blood pressure is not altered. Second, because prazosin treatment enhances skeletal muscle blood flow, tissue oxygenation was not compromised here. The same conclusion was reached by Dawson and Hudlicka (1989) when studying the effects of prazosin on skeletal muscle capillary density. Finally, the possibility that prazosin was directly stimulating fibroblast proliferation was addressed by studying the effects of prazosin on rat dermal fibroblasts in vitro. The results from the in vitro study indicate that fibroblast proliferation, as assessed with essentially the same immunolabeling technique for BRDU uptake used in the in vivo study, is not significantly influenced by the addition of prazosin to the culture media. Given the above, we conclude that the observed in vivo proliferative response is consistent with the hypothesis that increased hemodynamic stress stimulates the production of mitogenic growth factors which subsequently act on adventitial fibroblasts.
ACKNOWLEDGMENTS This work was supported by NIH HL-39680, HL-49146, and HL52309. R.J.P. is a Postdoctoral Fellow of the American Heart Association, Virginia Affiliate, Inc.
REFERENCES Dawson, J. L., and Hudlicka´, O. (1989). The effects of long term administration of prazosin on the microcirculation in skeletal muscles. Cardiovasc. Res. 23, 913 – 920.
Dusseau, J. W., and Hutchins, P. M. (1979). Stimulation of arteriolar number by salbutamol in spontaneously hypertensive rats. Am. J. Physiol. 236, H134 – H140. Hernandez, I., Cowley Jr., A. W., Lombard, J. H., and Greene, A. S. (1992). Salt intake and angiotensin II alter microvessel density in the cremaster muscle of normal rats. Am. J. Physiol. 263, H664 – H667. Hsieh, H. J., Li, N. Q., and Frangos, J. A. (1992). Shear-induced platelet derived growth factor gene expression in human endothelial cells is mediated by protein kinase C. J. Cell. Physiol. 150, 552 – 558. Osinski, M. A., Dahl, J. L., and Bass, P. (1993). Proliferation of mast cells in the smooth muscle of denervated rat jejunum. J. Autonomic Nervous Sys. 45, 164 – 174. Pearce, S. C., and Hudlicka, O. (1996). Possible involvement of prostaglandins in capillary growth in chronically stimulated skeletal muscles. Microcirculation 3, 181. [Abstract] Price, R. J., Owens, G. K., and Skalak, T. C. (1994). Immunohistochemical identification of arteriolar development using markers of smooth muscle differentiation: Evidence that capillary arterialization proceeds from terminal arterioles. Circ. Res. 75, 520 – 527. Price, R. J., and Skalak, T. C. (1996). Chronic a1 adrenergic blockade stimulates terminal and arcade arteriolar development. Am. J. Physiol. 271, H752 – H759. Price, R. J., and Skalak, T. C. (1995). A circumferential wall stressgrowth rule predicts arcade arteriole formation in a network model. Microcirculation 2, 41 – 51. Price, R. J., and Skalak, T. C. (1998). Distribution of Cellular Proliferation in Transverse Arterioles During Normal Maturation. Microcirculation. in press. Skalak, T. C., and Price, R. J. (1996). The Role of Mechanical Stresses in Microvascular Remodeling. Microcirculation 3, 143 – 165. Stingl, J., and Rhodin, J. A. G. (1994). Early postnatal growth of skeletal muscle blood vessels of the rat. Cell Tissue Res. 275, 419 – 434. Wilson, E., Mai, Q., Sudhir, K., Weiss, R. H., and Ives, H. E. (1993). Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J. Cell Biol. 123, 741 – 747. Ziada, A., Hudlicka, O., and Tyler, K. R. (1989). The effect of longterm administration of a1 blocker prazosin on capillary density in cardiac and skeletal muscle. Pflu¨gers Arch. 415, 355 – 360.
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Prazosin Administration Enhances Proliferation of Arteriolar Adventitial Fibroblasts Richard J. Price and Thomas C. Skalak Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia Received October 17, 1997
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 a1 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 õ8 mm diameter terminal arterioles and a 280% increase in the density of S-phase fibroblasts surrounding 8- to 12-mm 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. q 1998 Academic Press
Key Words: microcirculation; arteriolar development; angiogenesis; hemodynamic stress.
INTRODUCTION
New arterioles form in skeletal muscle when mesenchymal precursor cells on the abluminal surface of capillaries differentiate toward a smooth muscle phenotype (Stingl and Rhodin, 1994; Price et al., 1994). In previous studies, it was determined that both the rate of terminal arteriolar development (Price et al., 1994) and the density of proliferating cells surrounding terminal arteriole endings is greater in 4week-old weanling animals when compared to 9week-old juvenile animals (Price and Skalak, 1998). By eliminating the possibility that the proliferating adventitial cells were mast cells, it was concluded that these cells were most likely fibroblasts. Because rates of arteriolar development and fibroblast proliferation are correlated during maturation, these results are consistent with a role for fibroblast proliferation in the assembly of new terminal arterioles. We hypothesize that fibroblast proliferation, and perhaps migration to the arterializing capillary, precede the differentiation of these precursor cells into smooth muscle. Several previous studies indicated that elevated levels of hemodynamic stress in individual capillaries are capable of stimulating their transformation into new 0026-2862/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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terminal arterioles (Price and Skalak, 1994, 1996; Skalak and Price, 1996). For instance, terminal arteriolar development was enhanced and collateral vessel formation was increased in animals that were chronically vasodilated with the a1 adrenergic blocker prazosin (Price and Skalak, 1996). A similar result was obtained by Dusseau and Hutchins (1979) who observed an increase in the number of skeletal muscle terminal arterioles in animals that had received the b2 agonist salbutamol, a vasodilator. The objective of the current study was to test the hypothesis that chronic vasodilation with prazosin elicits enhanced proliferation of interstitial cells surrounding transverse arterioles. The density of S-phase cells surrounding small diameter terminal arterioles was significantly increased in animals treated with prazosin for 4 days, indicating that elevated levels of hemodynamic stress stimulate the proliferation of interstitial fibroblasts which may subsequently invest arterializing capillaries.
MATERIALS AND METHODS
(2) washing briefly in buffer, (3) incubating for 1 h at 377C in a 0.2 mg/ml solution of pepsin in 0.1 N HCL, and (4) incubating in 1 N HCL for 30 min at 377C. For two of the prazosin specimens, 1% Alcian blue was added to the incubating solution in step 3 above. Before starting the immunochemistry protocol, specimens were washed thoroughly in buffer. A stock solution of 0.4% (vol/vol) Triton X-100, 3% bovine serum albumin, and 5% (vol/vol) normal goat serum was used in each antibody incubation. Incubations lasted for 24 h and were done at room temperature. Concentrations are given in each step. Incubations proceeded in the following order: (1) Mouse monoclonal Ab to bromodeoxyuridine (1:40), (2) Goat anti-mouse IgG Fab fragments conjugated to fluorescein isothiocyanate (FITC, 1:100), (3) Hybridoma supernatant containing mouse monoclonal Ab to SM-MHC (1:1), and (4) Goat anti-mouse IgG Fab fragments conjugated to lissamine rhodamine sulfonyl chloride (LRSC, 1:100). The mouse monoclonal Ab to BRDU was from Dako Inc., the mouse monoclonal Ab to SM-MHC was developed at the University of Virginia hybridoma facility by Dr. Gary K. Owens, and the secondary Abs (Fab fragments) were from Jackson Immunoresearch Laboratories Inc.
Specimen Preparation Female Sprague – Dawley rats (n Å 5) received prazosin (50 mg/liter) in their drinking water for a period of 4 days. Treated and weight matched control animals (n Å 5) were anesthetized with an intramuscular injection of 13.3% urethane and 1% a-chloralose (0.6 ml/ 100 g). The right gracilis muscle was exposed by removing the overlying skin and fascia. Regions surrounding the gracilis muscle were packed in cotton to retain moisture. Bromodeoxyuridine (1005 M in Ringer’s solution) was administered to the muscle by superfusion at a flow rate of 4 ml/min. After 2 h of superfusion with the Ringer’s solution containing BRDU, a descending catheter was placed in the right saphenous artery. To fix the gracilis muscle in its in situ position, a solution of 3% paraformaldehyde in phosphate buffer was perfused through the saphenous artery catheter for 5 min. The muscle was then washed by perfusion with phosphate buffer. Muscles were dissected free and processed for immunochemistry by: (1) incubating in a 3 mg/ml solution of collagenase in buffer for 30 min at room temperature,
Measurements and Cell Identification After identifying TA trees with an LRSC filter setting and a low power (120) objective, a dual LRSC-FITC filter setting and a 163 objective were used to measure the length and outer diameter of each clearly visible TA segment and to identify S-phase adventitial cells. S-phase adventitial cells were defined as those cells that were both negative for SM-MHC and contained a BRDU-positive nucleus within 5 mm of the arteriolar outer wall. To determine if any S-phase adventitial cells were mast cells, 2 of the 5 prazosin specimens were counterstained with Alcian blue, which is capable of labeling mast cells in S-phase (Osinski et al., 1993). Sphase mast cells were, therefore, designated as those mast cells with an intracellular region of positive FITC labeling within 5 mm of the arteriolar outer wall. Proliferating smooth muscle and arteriolar endothelial cells were not included in this study because, consistent with an earlier study on cell proliferation during maturation (Price and Skalak, 1998), only a few were observed in Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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each group, and no meaningful conclusions could be made by counting these cell types. S-phase adventitial cell densities were calculated as the number of S-phase cells per unit surface area, as determined by the dimensions of the individual TA segments. S-phase cell densities were further reduced into two forms. In the first form, the density of S-phase cells per TA tree was calculated such that the n value was the total number of TA trees in each group. This data was compiled in a histogram format, and the mean value for the prazosin group was compared to the mean of the control group using a student’s t test at the P õ 0.05 level. In the second form, S-phase cell densities were determined for various diameter ranges throughout the TA trees. The following diameter ranges were chosen; õ8 mm, 8.1 – 12 mm, 12.1 – 16 mm, 16.1 – 20 mm, 20.1 – 24 mm, 24.1 – 28 mm, and ú28 mm. Here, n values were the total number of observed TA segments for each group. The means from each diameter range in the prazosin group were then compared to control for the same diameter range using an unpaired student’s t test with significance assessed at the P õ 0.05 level. For the Alcian blue counter stained specimens, the percentage of S-phase interstitial cells that were also mast cells was determined.
In Vitro Fibroblast Proliferation Assay To study the direct effects of prazosin on fibroblast DNA synthesis, fibroblasts were incubated in media containing prazosin and subsequently assayed for BRDU uptake. The in vitro protocol was designed to match, as closely as possible, the in vivo experimental conditions and immunoassay procedures. Rat fibroblasts were obtained by incubating skin explants in Dulbecco’s Modified Eagle Media containing 10% Fetal Bovine Serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 250 ng/ml amphotericin B for approximately 1 week. This period of time was sufficient for fibroblasts to migrate from the skin explant to the culture dish and become confluent in the region surrounding the explant. After removing the skin explants from the dish, fibroblasts were removed by trypsinization. Cultured fibroblasts were passaged three times in the media described above before being seeded into 35mm-diameter culture dishes at a concentration of 10,000 cells per dish. In preliminary experiments, this seeding
density was determined to yield approximately 80% confluency after 4 days. Because cell proliferation is inhibited when cells become confluent, a low seeding density was required to keep cells subconfluent during these studies. Experimental groups were: (1) media containing 0.5 mg/liter prazosin (n Å 10), (2) media containing 0.05 mg/liter prazosin (n Å 10), and (3) control media (n Å 10). Prazosin was dissolved in dimethylsulfoxyide (DMSO) before being added to culture media. Because the final vol/vol concentration of DMSO in prazosin treated media was 0.01%, 0.01% DMSO was added to control media as well. After 4 days, 1005 M BRDU was added to the culture media for 2 h. Media was then removed from the culture dishes and cells were washed in phosphate buffer before they were fixed in 3% paraformaldehyde for 5 min. A brief buffer wash was followed by incubations in 0.1 N HCl for 1 h and 1.0 N HCl for 30 min. Preliminary studies revealed that pepsin digestion was not required for the in vitro labeling. Antibody incubations were performed in the same stock solution as used in the in vivo assay. The first incubation was in the mouse monoclonal Ab to BRDU (1:100) for 2 h at room temperature. Following a wash in phosphate buffer, cells were then incubated for 1 h in goat anti-mouse IgG Fab fragments conjugated to FITC (1:200). For illustrative purposes, one dish from each group was also labeled for the intermediate filament vimentin using an antibody (clone V9 at a concentration of 1:100) that was directly conjugated to CY3. The vimentin Ab was from Sigma, Inc. To quantify fibroblast DNA synthesis, the total number of BRDU-positive nuclei per unit surface area was determined for each dish. Twenty randomly selected fields of view per dish were chosen for observation using an FITC filter and a 110 objective. After videotaping each field of view, the images were digitized and analyzed using the Optimas (v 6.1) software package. The total number of BRDU-positive nuclei per field of view was counted using the Optimas software and then scaled so that the reported result was the number of BRDU-positive nuclei per unit surface area. The mean number of BRDU-positive nuclei per dish was calculated for each group.
RESULTS On the day of the experiments, prazosin-treated and weight-matched control rats weighed ({SD) 216.8 {
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FIG. 1. Photomicrographic montage of a region of rat gracilis muscle from a prazosin-treated animal. (A) Transverse arteriole segment immunolabeled for SM-MHC with LRSC. (B) S-phase adventitial nucleus immunolabeled for BRDU with FITC. (C) Mast cells visualized under transmitted light with Alcian blue counterstaining. (D) Composite image of parts A, B, and C. Bar indicates 20 mm.
8.1 and 206.4 { 10.2 g, respectively. An example of the immunolabeling used in the in vivo studies is provided in Fig. 1. In Fig. 1A, a transverse arteriole that has been labeled for SM-MHC with a secondary Ab conjugated to LRSC is shown. The smooth muscle wrapping pattern is clearly evident and smooth muscle nuclei are visible as oblong or circular gaps in the SM-MHC labeling. Figure 1B depicts an S-phase cell that has been labeled for BRDU using antibodies conjugated to FITC. The punctate chromatin pattern is evident in this cell nucleus, which lies within 5 mm of the TA segment shown in Fig. 1A. Although S-phase interstitial cell nuclei were often oblong and parallel to the longitudinal axis of the nearby arteriole, such as in Fig. 1B, several shapes and orientations were observed. This particular specimen was also counterstained for mast cells using
Alcian blue. Three mast cells, shown in Fig. 1C, are evident in this region when viewed under transmitted light. In Figure 1D, the images from A, B, and C have been combined to illustrate the relative positions of the mast cells, S-phase nucleus, and TA segment. Note that the S-phase nucleus was not a mast cell nucleus. In fact, of the 125 nuclei within 5 mm of TA segments observed in the two Alcian blue counterstained specimens, none were mast cell nuclei. A few S-phase mast cells were, however, observed in other regions of the muscle. Figure 2 depicts the S-phase interstitial cell density data when defined as the number of S-phase interstitial cells per unit surface area of TA (cells/mm2). The mean density { SE was 34.97 { 3.04 (n Å 165 trees) for the prazosin group and 11.06 { 1.43 (n Å 160 trees) for the control group. These values are statistically different at Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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BRDU-positive nuclei per unit surface area for each of the three groups. No significant differences were found between groups, indicating that prazosin treatment for 4 days had no effect on fibroblast DNA synthesis.
DISCUSSION
FIG. 2. S-phase adventitial cell density histogram. Transverse arteriolar trees completely lacking S-phase adventitial cells are represented as the first bar along the horizontal axis (i.e., at the ‘‘0’’ tick). *Mean S-phase cell density per TA tree was significantly greater at the P õ 0.05 level for the prazosin group (n Å 165 TA trees) when compared to control (n Å 160 TA trees).
P õ 0.05. Figure 3 depicts S-phase interstitial cell densities throughout a range of TA outer diameters for both groups. For the control and prazosin groups, respectively, the õ8-mm range consists of 311 and 442 segments, the 8- to 12-mm range consists of 710 and 754 segments, the 12- to 16-mm range consists of 535 and 540 segments, the 16- to 20-mm range consists of 272 and 284 segments, the 20- to 24-mm range consists of 126 and 166 segments, the 24- to 28-mm range consists of 28 and 58 segments, and the ú28-mm range consists of 16 and 49 segments. The prazosin group exhibited a statistically significant eightfold increase from control in the density of S-phase cells surrounding TA segments less than 8 mm in diameter. In the 8- to 12-mm diameter range, a statistically significant 289% increase above control was observed for the prazosin group. No other significant differences were observed. Figure 4 illustrates the general appearance of the in vitro rat fibroblast cultures following immunolabeling for nuclear BRDU uptake using Abs conjugated to FITC. In Figs. 4A and 4B, respectively, S-phase nuclei are apparent in control cultured cells and cultured cells exposed to 0.5 mg/liter prazosin for 4 days. Immunolabeling for the intermediate filament vimentin using a CY3 conjugated Ab is shown in Figs. 4C and 4D for control and prazosin-treated cells, respectively, to illustrate that the fibroblasts were subconfluent after the 4day time period. Figure 5 depicts the mean number of
The objective of this study was to determine whether chronic vasodilation with the a1 adrenergic blocker prazosin enhances the proliferation of adventitial cells surrounding transverse arterioles. The density of S-phase interstitial cells within 5 mm of transverse arterioles was calculated using measurements taken from specimens that were dual-immunolabeled for SM-MHC and BRDU. Results indicated that the overall density of Sphase adventitial cells was increased in prazosintreated animals. This increase was primarily due to an increased density of cells surrounding small diameter (õ12 mm) terminal arterioles. Alcian blue counterstaining revealed that the proliferating cells were not mast cells, indicating by process of elimination that these cells were most likely fibroblasts. In vitro experiments demonstrated that prazosin treatment had no direct effect on fibroblast proliferation. Because prazosin treatment increases microvascular pressure and
FIG. 3. Bar graph of S-phase adventitial cell densities for various diameter ranges. Number of transverse arteriole segment observations (n) for control and prazosin groups, respectively, in each diameter range: õ8 mm, 311 and 442; 8 – 12 mm, 710 and 754; 12 – 16 mm, 535 and 540; 16 – 20 mm, 272 and 284; 20 – 24 mm, 126 and 166; 24 – 28 mm, 28 and 58; ú28 mm, 16 and 49. *Significantly different than control (same diameter range) at P õ 0.05.
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FIG. 4. Photomicrographs of immunofluorescently labeled rat dermal fibroblasts. (A and B) Control cultured fibroblasts (A) and fibroblasts exposed to 0.5 mg/liter prazosin for 4 days (B) labeled for BRDU uptake using a FITC conjugated secondary Ab. (C and D) Same regions of control and prazosin-treated cells as shown in (A and B), respectively, labeled for the intermediate filament vimentin using a primary Ab conjugated to CY3. After 4 days, cells were approximately 80% confluent. Bar indicates 30 mm.
flow, these results are consistent with the hypothesis that increased hemodynamic stress stimulates the proliferation of adventitial fibroblasts which may subsequently differentiate into smooth muscle. The current study was primarily undertaken to link the results of two previous studies performed in our laboratory. In the first study (Price and Skalak, 1996), prazosin treatment for 1 week stimulated the formation of new terminal arterioles in skeletal muscle. After 2
weeks of treatment, terminal arteriole development remained increased and a significant increase in the number of small diameter (õ15 mm) arcade arterioles was also observed. Similar results were obtained by Dusseau and Hutchins (1979) who observed that chronic administration of salbutamol, a b2 agonist and vasodilator, caused a 22% increase in the total number of small arterioles in Wistar – Kyoto rat cremaster muscle. In the second study (Price and Skalak, 1998), it was deterCopyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 5. Bar graph of in vitro S-phase cell densities for control fibroblasts, fibroblasts exposed to 0.5 mg/liter prazosin for 4 days, and fibroblasts exposed to 0.05 mg/liter prazosin for 4 days. No significant differences were observed between groups. n Å 10 dishes for each group.
mined that a relatively higher density of S-phase fibroblasts exists in the region surrounding terminal arterioles of 4-week-old weanling animals when compared to 9-week-old juvenile animals. Because the later result indicates that the density of S-phase adventitial fibroblasts correlates with the rate of arteriolar development during maturation, we hypothesized that fibroblast proliferation may be a component of capillary arterialization. Thus, the current study was designed to determine whether the correlation between increased Sphase adventitial fibroblast density and terminal arteriolar development is also present when prazosin is used as a stimulus for arteriolar growth. Indeed, the results clearly indicate that a population of transverse arteriolar adventitial cells, which are likely fibroblasts, proliferate in response to this treatment. Moreover, the results from this study are consistent with the previous study of S-phase adventitial cell proliferation (Price and Skalak, 1998) because enhanced proliferation of adventitial cells was greatest in the smallest diameter terminal arterioles. The local stimulus for adventitial cell proliferation generated by the prazosin treatment was most likely an increase in either wall shear stress or circumferential wall stress in the capillaries of treated rats. Dawson and Hudlicka (1989) and Ziada et al. (1989) have previously shown that capillary angiogenesis is stimulated in muscles that experience enhanced capillary blood flow in response to chronic treatment with pra-
zosin. Microvascular pressure is also elevated in capillaries and terminal arterioles of treated rats because, in a previous study (Price and Skalak, 1996), no change in systemic pressure was observed. The lack of a systemic pressure drop, in conjunction with vasodilation, indicates that the microvascular pressure gradient was shifted such that capillaries and terminal arterioles experienced greater pressures and circumferential wall stresses. Increased hemodynamic stresses (shear stress and stretch) are capable of triggering the production of mitogenic growth factors by the endothelium (Hsieh et al., 1992; Skalak and Price, 1996) and by smooth muscle (Wilson et al., 1993; Skalak and Price, 1996). In addition, Pearce and Hudlicka (1996) have observed an increase in the density of proliferating interstitial cells in skeletal muscle in response to electrical stimulation, an intervention that produces increased capillary wall shear stress and circumferential wall stress. Alterations in nitric oxide (NO) levels associated with changes in hemodynamics may also influence cell proliferation in this study. The potential effects of NO could be tested in the future using a NOS inhibitor. Although prazosin was chosen as the vasodilator for this particular study, alternative vasodilators such as nitrates, yohimbine, or hydralazine are good candidates for producing similar levels of hemodynamic stress and, potentially, arteriolar remodeling. While increased levels of hemodynamic stress appears to be the most likely explanation for the current results, there are potential confounding factors involved in this experiment. These factors include the effect of altered blood pressure on circulating levels of humoral factors, the effect of vasodilation on tissue oxygenation, and the direct effect of prazosin treatment on fibroblast proliferation. First, the effect of prazosin treatment on circulating ANG II, a mediator of microvascular growth and rarefaction (Hernandez et al., 1992), is unclear in this study because an inhibitor of ANGII, such as captopril, was not used. We have provided a detailed discussion of the potential role of ANG II in the prazosin treatment in an earlier study (Price and Skalak, 1996). Briefly, because decreased blood pressure is a stimulus for ANG II production and blood pressure throughout the experiment was unknown, the potential for ANG II production at various times during treatment clearly exists. However, in the earlier study (Price and Skalak, 1996), no significant decrease in
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blood pressure was observed after 7 days, indicating that when this particular dosage is provided for short periods of time, blood pressure is not altered. Second, because prazosin treatment enhances skeletal muscle blood flow, tissue oxygenation was not compromised here. The same conclusion was reached by Dawson and Hudlicka (1989) when studying the effects of prazosin on skeletal muscle capillary density. Finally, the possibility that prazosin was directly stimulating fibroblast proliferation was addressed by studying the effects of prazosin on rat dermal fibroblasts in vitro. The results from the in vitro study indicate that fibroblast proliferation, as assessed with essentially the same immunolabeling technique for BRDU uptake used in the in vivo study, is not significantly influenced by the addition of prazosin to the culture media. Given the above, we conclude that the observed in vivo proliferative response is consistent with the hypothesis that increased hemodynamic stress stimulates the production of mitogenic growth factors which subsequently act on adventitial fibroblasts.
ACKNOWLEDGMENTS This work was supported by NIH HL-39680, HL-49146, and HL52309. R.J.P. is a Postdoctoral Fellow of the American Heart Association, Virginia Affiliate, Inc.
REFERENCES Dawson, J. L., and Hudlicka´, O. (1989). The effects of long term administration of prazosin on the microcirculation in skeletal muscles. Cardiovasc. Res. 23, 913 – 920.
Dusseau, J. W., and Hutchins, P. M. (1979). Stimulation of arteriolar number by salbutamol in spontaneously hypertensive rats. Am. J. Physiol. 236, H134 – H140. Hernandez, I., Cowley Jr., A. W., Lombard, J. H., and Greene, A. S. (1992). Salt intake and angiotensin II alter microvessel density in the cremaster muscle of normal rats. Am. J. Physiol. 263, H664 – H667. Hsieh, H. J., Li, N. Q., and Frangos, J. A. (1992). Shear-induced platelet derived growth factor gene expression in human endothelial cells is mediated by protein kinase C. J. Cell. Physiol. 150, 552 – 558. Osinski, M. A., Dahl, J. L., and Bass, P. (1993). Proliferation of mast cells in the smooth muscle of denervated rat jejunum. J. Autonomic Nervous Sys. 45, 164 – 174. Pearce, S. C., and Hudlicka, O. (1996). Possible involvement of prostaglandins in capillary growth in chronically stimulated skeletal muscles. Microcirculation 3, 181. [Abstract] Price, R. J., Owens, G. K., and Skalak, T. C. (1994). Immunohistochemical identification of arteriolar development using markers of smooth muscle differentiation: Evidence that capillary arterialization proceeds from terminal arterioles. Circ. Res. 75, 520 – 527. Price, R. J., and Skalak, T. C. (1996). Chronic a1 adrenergic blockade stimulates terminal and arcade arteriolar development. Am. J. Physiol. 271, H752 – H759. Price, R. J., and Skalak, T. C. (1995). A circumferential wall stressgrowth rule predicts arcade arteriole formation in a network model. Microcirculation 2, 41 – 51. Price, R. J., and Skalak, T. C. (1998). Distribution of Cellular Proliferation in Transverse Arterioles During Normal Maturation. Microcirculation. in press. Skalak, T. C., and Price, R. J. (1996). The Role of Mechanical Stresses in Microvascular Remodeling. Microcirculation 3, 143 – 165. Stingl, J., and Rhodin, J. A. G. (1994). Early postnatal growth of skeletal muscle blood vessels of the rat. Cell Tissue Res. 275, 419 – 434. Wilson, E., Mai, Q., Sudhir, K., Weiss, R. H., and Ives, H. E. (1993). Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J. Cell Biol. 123, 741 – 747. Ziada, A., Hudlicka, O., and Tyler, K. R. (1989). The effect of longterm administration of a1 blocker prazosin on capillary density in cardiac and skeletal muscle. Pflu¨gers Arch. 415, 355 – 360.
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