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Am J Physiol Heart Circ Physiol 293: H2429-H2437, 2007. First published July 20, 2007; doi:10.1152/ajpheart.00100.2007
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Involvement of MMPs in the outward remodeling of collateral mesenteric arteries

Tara L. Haas,1 Jennifer L. Doyle,1 Matthew R. Distasi,3 Laura E. Norton,2 Kevin M. Sheridan,2 and Joseph L. Unthank2,3

1School of Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada; and Departments of 2Surgery and 3Cellular and Integrative Physiology, Indiana University Medical Center, Indianapolis, Indiana

Submitted 24 January 2007 ; accepted in final form 13 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Persistent elevation in shear stress within conduit or resistance arteries causes structural luminal expansion, which serves to normalize shear stress while maintaining increased flow to the downstream vasculature. Although it is known that this adaptation involves cellular proliferation and remodeling of the extracellular matrix, the specific cellular events underlying these responses are poorly understood. Matrix metalloproteinases (MMPs) contribute to extensive remodeling of the extracellular matrix in conduit vessels and vein grafts exposed to high flow. However, involvement of MMPs in remodeling of small muscular collateral arteries, which are exposed to less severe increases in shear stress, has not been tested. We utilized an established model of outward remodeling in mesenteric collateral arteries to determine whether MMPs were upregulated during the remodeling response and to test whether MMP activity was required for luminal expansion. By 4 days, MMP-2 and membrane type 1 MMP (MT1-MMP), but not MMP-9, protein levels were significantly elevated in collateral arteries, as assessed by gelatin zymography and immunostaining. MMP-2 and MT1-MMP proteins, together with their respective transcriptional activators c-Jun and Egr-1 were localized predominantly to the smooth muscle layer of the collateral arteries. The general MMP inhibitor doxycycline prevented luminal expansion of collateral arteries but did not affect the endothelial cell proliferative or medial growth responses. In conclusion, this study provides evidence that MMP-2 and MT1-MMP are upregulated in collateral arteries exposed to elevated shear stress and that MMP activity is essential for the full remodeling response that leads to outward luminal expansion.

matrix metalloproteinase; collateral artery; shear stress

VASCULAR SHEAR STRESS plays a central role in regulating vascular tone and maintaining blood vessel structure. Endothelial cells act as the primary flow sensors within the vessel wall, because they have direct contact with plasma and the capacity, through a variety of cell surface receptors, to transduce intracellular signals in response to alterations in shear stress (31). Acutely, an elevation in the magnitude of shear stress leads to increased cytoplasmic calcium, activation of endothelial nitric oxide (NO) synthase and cyclooxygenase, and generation of vasodilators such as NO, endothelium-derived hyperpolarizing factor, and prostaglandin E2, which act to dilate the vessel and normalize shear stress. Conversely, reduced shear stress results in decreased production of these vasodilatory substances, with subsequent vessel constriction.

Persistent alteration of shear stress results in structural modification of blood vessels. Luminal expansion occurs in response to prolonged elevation of shear stress, whereas sustained attenuation of shear stress is associated with luminal reduction of conduit and resistance arteries (6, 27, 40, 55, 60). These diameter changes proceed until wall shear rate is normalized (27, 55, 60). In the resistance vasculature, this shear- or flow-mediated regulation is believed to be a mechanism to match tissue perfusion to metabolic need (45). The pathophysiological relevance of such a mechanism is seen in the luminal expansion of collateral arteries distal to an occlusion and the regression of luminal diameter when flow is chronically reduced, as with muscle disuse. The former acts in a compensatory manner to enhance tissue perfusion, whereas the latter may lead to further pathology and limit skeletal muscle perfusion and exercise tolerance (46).

Cellular proliferation and reorganization of extracellular matrix components are required to generate a larger lumen. Endothelial cell proliferation precedes luminal expansion in response to elevated shear level in conduit and resistance arteries (34, 54, 57). In small muscular arteries and arterioles, luminal expansion also involves structural modifications within the medial and adventitial layers (40, 54, 57). Matrix metalloproteinases (MMPs) constitute a family of zinc- and calcium-dependent proteases capable of cleaving numerous components of the extracellular matrix, cell surface receptors, and matrix-bound growth factors. MMP activity commonly is a factor underlying the structural remodeling of tissue. MMP-2, MMP-9, and membrane type 1 MMP (MT1-MMP), in particular, are associated with vascular remodeling. Most knowledge of the role of MMPs in flow-induced vascular remodeling is based on models with vein grafts or large elastic arteries in which shear rates are dramatically elevated and MMP expression may be 10–100 times control values (44). Little is known about the participation of MMPs in the remodeling that occurs in response to more physiological levels of altered shear and, specifically, in small muscular arteries. In apparent contradiction to data gathered from large artery remodeling studies, exposure to elevated shear stress significantly downregulates expression of MT1-MMP and MMP-2 in cultured microvascular endothelial cells (36, 59). The objective of the present study was to investigate the hypothesis that moderately elevated flow/shear in small resistance vessels is sufficient to activate MMPs and that this activation is required for the outward remodeling of these vessels. To accomplish this objective, we utilized an established model of outward remodeling in mesenteric collateral arteries. Previous studies with this model in young normotensive rats showed that arterial ligation elevates wall shear rate 100–200% in the adjacent collaterals (52–55). These collateral vessels begin to enlarge within 2–3 days, and luminal dimensions are increased ~35% and 65% within 7 and 28 days, respectively (55, 57).


   METHODS
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 METHODS
RESULTS
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Experimental model. Animal procedures were approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine. Ileal arteries of male Wistar rats (~200 g body wt; Harlan Sprague Dawley, Indianapolis, IN) were surgically ligated to produce a collateral-dependent region, as previously described (55, 57). Briefly, laparotomy was performed with aseptic technique, and the terminal ileum was exteriorized into a heated tissue support chamber positioned on the abdomen of the rat. At all times, the tissues were immersed in PBS. Sequential ileal arteries were ligated with 9-0 monofilament, such that a segment of terminal ileum was made dependent on blood flow through a preexisting collateral artery on each end of the ligated region (see schematic in Ref. 55). The bowel segment was selected so that there were ~50 first-order arterioles (on 1 side of the bowel) between collaterals, and, typically, 3 but no more than 4 arteries were ligated. The bowel was then returned to the abdominal cavity, and the incision was closed in two layers. Animals were allowed to recover, and final experiments were performed 2–7 days later.

Gelatin zymography. The mesenteric vasculature was perfused with 60 ml of cold PBS via a caudal aorta cannula. The perfusion was completed after ligation of the renal arteries and the proximal abdominal aorta immediately below the diaphragm. The terminal ileum and mesentery were excised and placed on a frozen silicone disk. With the silicone disk on a slab of dry ice, the control and collateral arteries were isolated, excised, and snap frozen in liquid nitrogen. Frozen arteries were pulverized in the presence of liquid nitrogen and then lysed in 50 µl of 0.1 M Tris buffer (pH 8.7) containing 0.01% Triton X-100. Samples were quantified for total protein using a micro-bicinchoninic acid assay according to the manufacturer's directions (Pierce). Ten micrograms of total protein per sample were loaded onto 8% SDS-PAGE gels impregnated with 0.1% gelatin and separated using nondenaturing electrophoresis. A gelatin zymography standard, consisting of recombinant MMP-9 and MMP-2 proteins in latent and active forms (Chemicon), was loaded on each gel to provide a positive control and appropriate size markers. Gel development was carried out as previously described (15). Gels were imaged on a gel documentation system (FluorChem, AlphaInnotech) using white light transillumination, and band intensities were quantified using AlphaEase software (AlphaInnotech). For MMP-2, total protein was calculated as the sum of band intensities for latent and active forms. These values were normalized to that of the first sham condition on each gel. For percent active MMP-2, intensity of the active (62-kDa) band was expressed as a percentage of total MMP-2. Where detectable, intensities of bands corresponding to pro-MMP-9 and the high-molecular-weight dimerized form of pro-MMP-9 also were quantified. Relative amount of active MMP-2 was calculated for the doxycycline (DOX) experiment. The band intensities of 62-kDa MMP-2 were normalized to that of the control untreated arteries. Statistically significant differences were tested by one-way ANOVA followed by Tukey's post hoc tests (P < 0.05).

Immunofluorescence. Control and collateral arteries were removed from rats 4 days after surgery, embedded in optimal cutting temperature medium, and frozen at –80°C. Blocks were sectioned to generate 10-µm cross sections of the arteries, which were blocked in 5% normal goat serum (Sigma) and 0.01% Triton X-100 in PBS and then incubated for 1 h in primary antibody diluted in blocking solution. Antibody sources and dilutions were as follows: anti-MMP-2 (Ab 809), anti-MMP-9 (MAb 3309), and anti-MT1-MMP (Ab 815), each diluted 1:200 (Chemicon); anti-Egr-1, diluted 1:200 (Santa Cruz); and anti-phosphorylated c-Jun, diluted 1:500 (Cell Signaling). The sections were washed and then incubated for 1 h in secondary antibody (goat anti-rabbit or anti-mouse Alexa Fluor 568, Molecular Probes). The sections were washed again and counterstained with nuclear stain (diamidino-2-phenylindole; Molecular Probes), and coverslips were applied using ImmunoFluor mounting medium (ICN). Sections were viewed on a fluorescence microscope (Axiovert 200, Zeiss) using a x40 F Fluar oil immersion objective (NA 1.3). Images were captured using a digital cooled charge-coupled device camera (Quantix 57, Photometrics) controlled by Metamorph software (Universal Imaging). For each immunostaining protocol, exposure times were identical for control and collateral arteries. Results were analyzed only on those sections in which a lumen and endothelial and smooth muscle cell layers were visible. Metamorph software was used to calculate average pixel intensities at five to seven independent locations per cross section. Locations immediately adjacent to the artery were used for calculation of background pixel intensities for each cross section. Background-subtracted signals were averaged using a minimum of five independent cross sections from a total of three different rats per condition. Values are means ± SE. Statistical significance was assessed using two-tailed unpaired Student's t-tests, with significance established as P < 0.05.

MMP inhibition and measurement of arterial dimensions. Some animals were treated with DOX (30 mg·kg–1·day–1 in drinking water) beginning 3 days before surgery. DOX acts as a nonspecific MMP inhibitor (4, 25, 29, 30, 32). In vivo maximally dilated inner diameters (obtained by superfusion with 0.1 mM adenosine and 0.01 mM sodium nitroprusside) were measured in control and collateral arteries from untreated and DOX-treated rats immediately after arterial ligation and again 7 days later. After imaging on day 7, the caudal aorta was cannulated and the mesenteric circulation was perfusion fixed with 4% paraformaldehyde at the animal's mean arterial pressure. Control and collateral arteries then were excised and processed for plastic embedding and subsequent histological examination, as described previously (57). Vessel cross-sectional areas were traced using Metamorph software for quantification of arterial dimensions. Three sections were averaged for each vessel, and the measurements for each artery class (control or collateral) were averaged for each animal. Differences in arterial growth (percent change in diameter between observations), intimal cell number, and medial area and thickness were assessed with two-way ANOVA, and multiple pairwise comparisons were performed with the Holm-Sidak method.


   RESULTS
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DISCUSSION
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Control and collateral (high-flow) arteries were analyzed by gelatin zymography to examine the time course of MMP-2 and MMP-9 production and activation in response to altered blood flow (Fig. 1A). MMP-2 was produced constitutively at low levels in sham-operated animals. Total MMP-2, a measure of the overall amount of MMP-2 protein produced in the cell, inasmuch as it accounts for protein in the latent (72-kDa) and active (62-kDa) forms, increased 1.8-fold after 4 days and remained significantly elevated at 7 days (Fig. 1B). A change in the amount of active MMP-2 provides a sensitive indicator of alterations in the amount of cell surface MT1-MMP, which is the primary physiological activator of MMP-2 (15, 28). The percentage of MMP-2 in the active (62-kDa) form also increased significantly after 4 days (Fig. 1C). MMP-9 was not detectable in arteries from sham-operated animals. The latent (92-kDa) form of MMP-9 was detectable in some control and collateral arteries, but this response was variable among the rats, and there was no difference between control and collateral groups (P = 0.18; Fig. 1D). The active (82-kDa) form of MMP-9 was not detectable in any samples. A high-molecular-weight band, likely corresponding to the dimerized form of latent MMP-9, was detectable in some control and collateral arteries (Fig. 1A). The intensity of this band tended to increase with time after ligation in control and collateral vessels, although this trend was not significant (P = 0.25; Fig. 1E).


Figure 1
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  		Fig. 1. Latent and active forms of matrix metalloproteinase (MMP)-2 protein increase significantly during flow remodeling response. A: representative gelatin zymographs. MMP-2 is present in latent (72-kDa) and active (62-kDa) forms; MMP-9 is present in latent (92-kDa) form and in high-molecular-weight form corresponding to dimerized latent MMP-9 (*). Arteries were analyzed 2, 4, or 7 days after ligation surgery. Purified MMP-9 and MMP-2 proteins (Std) were used to validate identification of gelatinolytic bands. S, sham operated; C, control artery; E, experimental collateral artery. B: densitometric analysis of MMP-2 protein 2, 4, and 7 days after ligation surgery. Total MMP-2 protein was significantly increased in collateral compared with time-matched control arteries at 4 and 7 days. Total MMP-2 was calculated using summed intensities of latent and active bands for each condition, which then were normalized to sham-operated condition. *P < 0.05 vs. time-matched control (n = 4). C: percentage of MMP-2 in active (62-kDa) form 2, 4, and 7 days after ligation surgery. Percentage of active MMP-2 increased significantly compared with control arteries at 4 days. *P < 0.05 vs. control at 4 days (n = 4). This time point corresponds to the greatest rate of luminal expansion in mesenteric collateral arteries after abrupt arterial occlusion (55). D: amount of MMP-9 quantified by densitometry. Not all samples showed detectable levels of MMP-9. When detected, MMP-9 was observed in the latent (92-kDa) form only. MMP-9 was not significantly elevated in collateral compared with control arteries at any time point. E: amount of high-molecular-weight MMP-9 quantified by densitometry. Not all samples showed detectable levels of this form of MMP. Although a trend existed for increased amounts of this MMP at later time points, there was no difference between control and collateral arteries at any time point.

 
Protein distribution of MMP-2 and its activator MT1-MMP was examined by immunofluorescence of mesenteric arteries subjected to 4 days of high flow or time-matched controls. MMP-2 and MT1-MMP immunoreactivity was detectable but faint in control arteries (Fig. 2, A and D, respectively). In collateral arteries, immunoreactivity of MMP-2 (Fig. 2B) and MT1-MMP (Fig. 2E) was intense and appeared localized predominantly in the smooth muscle layers, with minimal expression in the endothelium and adventitia. Results of pixel intensity quantification of the immunofluorescent signals are summarized in Fig. 2, C and F. In contrast, MMP-9 immunoreactivity was minimal in control and collateral arteries (see supplemental Fig. 1 in the online version of this article at the American Journal of Physiology-Heart and Circulatory Physiology website).


Figure 2
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  		Fig. 2. Immunolocalization of MMP-2 and MT1-MMP in control and collateral arteries. Representative images are shown for control (A and D) and collateral (B and E) arteries immunostained for MMP-2 (A and B) or MT1-MMP (D and E) 4 days after ligation. Sections were counterstained with diamidino-2-phenylindole (DAPI, blue) to visualize nuclei. A: diffuse staining of MMP-2 within smooth muscle layers of control arteries. B: intense staining of MMP-2 in collateral arteries. Immunostaining appeared predominantly in smooth muscle layers, with intermittent endothelial staining. D: similar pattern of weak staining of MT1-MMP, the activator of MMP-2, in control arteries. E: in collateral arteries, localization of MT1-MMP, with intense staining, to smooth muscle layer, with intermittent labeling of endothelial cell layer and adventitia. Arrows, staining within smooth muscle layers; solid arrowheads, endothelial cell staining; open arrowheads, adventitial cells. L, lumen. Magnification x400. C and F: pixel intensities [arbitrary units (AU)] for MMP-2 and MT1-MMP. *P < 0.05 vs. control.

 
The transcription factors c-Jun and Egr-1 are known to activate mechanical stress-induced transcription of MMP-2 and MT1-MMP, respectively (16, 23). Consistent with the observed increase in MMP-2 and MT1-MMP, immunofluorescent detection of these transcription factors showed intense staining in collateral arteries at day 4 (Fig. 3, B and E) compared with faint signals in the control arteries (Fig. 3, A and D). Phosphorylated c-Jun (indicative of activated c-Jun) localized predominantly to nuclei of smooth muscle cells within the collateral artery (Fig. 3B), with some positive staining in nuclei lining the lumen. Egr-1 immunoreactivity was visible in smooth muscle of control arteries (Fig. 3D) but was noticeably present in a greater portion of the cells and at higher intensity in experimental collateral arteries (Fig. 3E). Quantification of phosphorylated c-Jun and Egr-1 intensities is summarized in Fig. 3, C and F, respectively.


Figure 3
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  		Fig. 3. Immunodetection of phosphorylated c-Jun and Egr-1 in control and collateral arteries. Representative images are shown for phosphorylated c-Jun (A and B) or Egr-1 (D and E) immunostaining of control (A and D) and collateral (B and E) arteries 4 days after ligation surgery. Sections were counterstained with DAPI (blue) to visualize nuclei. A: occasional phosphorylated c-Jun staining within nuclei of control arteries. B: intense labeling with phosphorylated c-Jun antibody of nuclei of smooth muscle cells within collateral arteries, as seen by the many green-blue nuclei (indicating overlapping phosphorylated c-Jun and DAPI fluorescence). D: patchy immunoreactivity for Egr-1 within smooth muscle layers of control arteries. E: localization of Egr-1, with intense staining throughout smooth muscle cell layer of collateral arteries. Some cells of endothelial layer and adventitia also were positive for Egr-1. Magnification x400. C and F: pixel intensities for phosphorylated c-Jun and Egr-1. *P < 0.05 vs. control.

 
Inhibition of MMP production and activity can be achieved with the tetracycline antibiotic DOX. Gelatin zymography was performed to determine the effects of DOX treatment on MMP-2 levels. Total MMP-2 protein levels decreased significantly in control and collateral arteries of rats treated with DOX (Fig. 4A). A significant difference in the relative levels of the active (62-kDa) band was observed between DOX- and vehicle-treated groups (Fig. 4B). Treatment of rats with DOX resulted in significant attenuation of the remodeling response at day 7 (Fig. 5). Collateral arteries in untreated rats (Fig. 5B) showed a typical pattern of luminal expansion compared with arteries subjected to normal flow (Fig. 5A). Morphology of normal-flow arteries from DOX-treated rats (Fig. 5C) was similar to that of untreated arteries. However, DOX-treated collateral arteries did not exhibit luminal expansion in response to the elevated flow (Fig. 5D). In vivo measurements of artery inner diameters provided quantification of the remodeling response in untreated and DOX-treated collateral arteries (Fig. 6A). Interestingly, intimal proliferation was not inhibited by DOX treatment, inasmuch as the number of intimal nuclei in arterial cross sections was similar for collateral arteries from untreated and DOX-treated animals (Fig. 6B). Medial cross-sectional area and medial thickness increased significantly in collateral compared with control arteries, and these responses also were not attenuated by DOX treatment (Fig. 6C).


Figure 4
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  		Fig. 4. Doxycycline (DOX) reduces MMP-2 levels. Control and collateral arteries from untreated and DOX-treated rats (4 days after ligation) were analyzed by gelatin zymography. A: densitometric analysis of total MMP-2 (sum intensities of 72- and 62-kDa bands) in control and collateral arteries from untreated and DOX-treated rats. B: relative amount of active (62-kDa) MMP-2 increased in collateral arteries from untreated animals but failed to increase in DOX-treated collateral arteries. *P < 0.05 vs. untreated control (n = 3). #P < 0.05 vs. untreated.

 

Figure 5
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  		Fig. 5. DOX treatment of control and collateral arteries. Representative images are shown for perfusion-fixed, plastic-embedded cross sections of control and collateral arteries 7 days after ligation surgery. A and B: control and collateral arteries, respectively, from untreated animals. C and D: control and collateral arteries, respectively, from DOX-treated animals. Sections were stained with hematoxylin and eosin. Magnification x100. Scale bar, 100 µm.

 

Figure 6
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  		Fig. 6. DOX inhibits luminal expansion in collateral arteries but does not prevent intimal or medial growth. A: quantification of artery remodeling showed significant luminal expansion in untreated collateral arteries compared with control arteries. *P < 0.05 vs. untreated control (n = 4). DOX-treated collateral arteries did not undergo significant luminal expansion compared with control arteries. Percent change in luminal diameter from immediately to 7 days after ligation was calculated from luminal diameters measured by in vivo video microscopy under maximally dilated conditions before fixation. There was no difference in initial diameter of control or collateral arteries in untreated or DOX-treated groups, and average diameter was 333 ± 6 µm. Diameter was increased significantly only in untreated collateral arteries (21%, P < 0.01, n = 4). B: number of intimal nuclei per cross section increased in collateral arteries of untreated and DOX-treated animals. *P < 0.01 vs. control. No significant difference was observed between untreated and DOX-treated samples. C: medial cross-sectional areas and medial thicknesses were increased significantly in collateral vs. control arteries from untreated and DOX-treated groups. *P < 0.05 (n = 4). Medial thickness was decreased significantly in DOX-treated compared with untreated control arteries. #P = 0.04 (n = 4). Neither medial cross-sectional area nor medial thickness was significantly reduced in DOX-treated compared with untreated collateral arteries (P > 0.05, n = 4).

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study provides the first measure of MMPs in small muscular arteries undergoing collateralization within a clearly defined collateral path and of a size typical of peripheral collateral vessels (20, 33, 42, 56) and under conditions in which the hemodynamic stimuli are known (55). Although the present study confirms that activity of MMPs is critical for the flow-induced outward artery remodeling that is associated with luminal expansion, it also reveals significant differences compared with earlier studies. This work provides evidence that production of MMP-2 and MT1-MMP, but not MMP-9, increases in collateral arteries subjected to a ~100–200% elevation in wall shear stress (52–55). The substantial increase in production of these MMPs occurs predominantly in the smooth muscle layer of the collateral arteries, in the absence of visible fragmentation of the internal elastic lamina and without formation of a neointima.

Previous reports of MMPs in collateral vessels are limited to vessels in the canine heart after occlusion of the canine circumflex left coronary artery. MMP-2 was expressed at high levels in the media of these early-growing collateral vessels, which correlated with fragmentation of the internal elastic lamina and reduced detection of smooth muscle cell basement membrane (10). Such fragmentation was not observed in mesenteric collateral arteries in the present study (Fig. 5) or previous studies (54, 57). Other reports shown that expression of MMP-2 and MMP-9 during coronary arteriogenesis was primarily elevated in the neointima and adventitia (8, 9). In some earlier studies of vessels undergoing remodeling distal to an occlusion site, their position in the collateral path was unclear and the magnitude of alteration in hemodynamic stimuli was unknown. In contrast, the moderate level of flow elevation in mesenteric arteries induced by adjacent arterial ligation elevates perivascular NO levels (22), which is associated with promotion of anti-inflammatory signals (58). This is consistent with the premise that the nature of the remodeling response depends on stimulus magnitude and may explain why a neointima does not develop in this model of collateral growth.

MMP-2 and MMP-9 have been identified as key metalloproteinases involved in flow-mediated remodeling in large vessels (44, 51). MMP-2 is the primary MMP upregulated by chronic shear elevation in conduit arteries (44, 51). However, the relationship between MMP-2 production and shear stress may not be linear, because MMP-2 also has been reported to increase in response to decreases in shear stress in vein grafts and large arteries (5).

In the present study, zymography showed no evidence of MMP-9 in many arteries, whereas 92-kDa (monomeric) and ~210-kDa (dimeric) forms of MMP-9 were detectable in several arteries. There were no differences in MMP-9 protein level between control and collateral arteries. MMP-9, the dominant MMP involved in remodeling of vascular grafts and angiogenesis induced by severe hindlimb ischemia (femoral artery and vein excision), is secreted primarily by inflammatory cells (26). Although it is clear that inflammation can promote angiogenesis and collateral growth and the involvement of inflammatory cells and MMP-9 is considered by some to be essential for collateral growth (19, 43), there also is evidence that vascular remodeling, including collateral growth, can occur with limited, if any, inflammation. Using gradual arterial occlusion, Tang et al. (49) showed significant vascular compensation in the rat hindlimb, even though inflammatory cell infiltration was not apparent. Furthermore, even studies that report a critical or essential role of inflammatory cells in vascular compensation demonstrate significant compensation when circulating and tissue levels of inflammatory cells are reduced (17, 18). In our mesenteric model, we have not observed histological evidence of inflammatory cell infiltration within the collateral wall (57). This result, together with the lack of elevated MMP-9 activity in collateral arteries, suggests that inflammatory cell infiltration is not involved in the outward remodeling of these vessels. The nature and magnitude of the shear stimulus appear to determine the extent of involvement of inflammatory cells (2, 49, 54).

Although elevated pressure, or strain, is a strong inducer of MMP-2 and MMP-9 production in smooth muscle cells (1), we consider that induction of MMP by elevated circumferential wall strain is unlikely to be involved in collateral vessel remodeling. As a result of arterial occlusion, luminal pressure is decreased along the collateral pathway. Measurements in our model have shown arterial pressure to be unchanged at the beginning of the collateral pathway but decreased to ~30% of mean arterial pressure at the distal end (52, 55). This distal drop in pressure increases the pressure gradient for flow through the collateral vessels and also decreases the collateral transmural pressure. The midzone and reentry (distal) portions of the collateral path, which experience the decrease in transmural pressure, are the sites of greatest luminal enlargement (33).

The cellular signals underlying the upregulation of MMP-2 and MT1-MMP in the collateral arteries have not been identified. Increased levels of phosphorylated c-Jun and Egr-1 (known to promote transcription of MMP-2 and MT1-MMP, respectively) suggest that the upregulation of these proteases occurs via increased transcription. NO inhibits MMP-2 production in endothelial and smooth muscle cells (14, 35, 36), and capillary endothelial cells downregulate MMP-2 production in response to elevated shear stress (36). Thus it is improbable that shear stress-induced production of NO causes the resultant increase in MMPs in the collateral arteries. On the other hand, shear stress-induced production of vasoactive substances, such as prostaglandins and/or endothelium-derived hyperpolarizing factor, contributes to flow-mediated dilation responses (47) and may participate in the longer-term remodeling through regulation of MMPs. In support of this possibility, prostaglandins E2 and F{alpha} were shown previously to be associated with increased expression and activation of MMP-2 in a variety of cell types (12, 21, 38, 48).

DOX has been used successfully to inhibit aneurysm formation and to reduce inward remodeling of carotid arteries (39, 50), and this effect has been attributed primarily to the reduced production and activity of MMP-9 and MMP-2 in smooth muscle (13). Similarly, we observed a decrease in the total level of MMP-2 and the amount of active MMP-2 in arteries from DOX-treated animals. MMP-2 and MT1-MMP act collaboratively to promote matrix protein degradation and cell movement (24, 37). Use of the broad-spectrum inhibitor of MMPs does not permit conclusion on the roles of individual MMPs in collateral artery remodeling. It is important to note that the functional significance of these MMPs in collateral artery remodeling also may rely on the effective modulation of additional proteolytic pathways. For example, urokinase plasminogen activator was reported to play a role in ischemia-induced arteriogenesis (11), and it was postulated that activation of urokinase plasminogen activator can occur via cell surface-localized active MMP-2 (41). Tissue transglutaminase activity, also involved in artery remodeling (2), is subject to regulation via proteolytic cleavage by MMP-2 (3).

In the present study, DOX impaired luminal expansion but did not affect the increased endothelial cell proliferation or the increased medial area and medial thickness in collateral arteries. Earlier works have shown that endothelial cell proliferation precedes artery enlargement in flow-stimulated carotid (34) and mesenteric arteries (54). Our present data support the hypothesis that endothelial cell proliferation is an early event in the remodeling process and does not depend on MMP activity. The increased medial thickness of collateral arteries 1 wk after ligation is postulated to depend on smooth muscle cell hypertrophy and/or matrix accumulation (57), and smooth muscle cell proliferation also may contribute to this process (7).

In summary, these results provide further insight into the process of small artery enlargement. In the absence of MMP activity, the proliferative/hypertrophic signals activated by increased wall shear rate are insufficient to permit structural expansion of luminal diameter. Interestingly, the phenotype observed in collateral arteries of DOX-treated animals is comparable to that in a previous study in which the collateral arteries of aged rats underwent medial hypertrophy and intimal proliferation but had substantially impaired luminal expansion. When considered together with the present study, these results indicate the value of continued investigation into the roles that abnormalities in MMP regulation and matrix proteolysis may play in impaired collateral growth.


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 GRANTS
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This project was supported by National Heart, Lung, and Blood Institute Grants HL-42898 (J. L. Unthank) and Canadian Institutes of Health Research Grant MOP-94546 (T. L. Haas).


   ACKNOWLEDGMENTS
 
We appreciate the expert histological assistance of Jennifer Stashevsky and the helpful advice of Dr. Sandra Davidge on zymography analysis of arteries.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Haas, School of Kinesiology and Health Sciences, Rm. 341 Farquharson, York Univ., 4700 Keele St., Toronto, ON Canada M3J 1P3 (e-mail: thaas{at}yorku.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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