β1-Integrin Is Essential for Vasoregulation and Smooth Muscle Survival In VivoSignificance
Objective—Integrins contribute to vascular morphogenesis through regulation of adhesion and assembly of the extracellular matrix. However, the role of β1-integrin in the mature vascular wall is less clear.
Approach and Results—We sought to determine the function of β1-integrin in mature smooth muscle cells in vivo using a loss of function approach by crossing a tamoxifen-inducible sm22αCre line to a floxed β1-integrin transgenic line. Adult mice lacking smooth muscle β1-integrin survived only 10 weeks post induction. The deletion of β1-integrin resulted in profound loss of vasomotor control. Histological analysis revealed progressive fibrosis in arteries with associated apoptosis of smooth muscle cells, which was not rescued by adventitial stem cells. Smooth muscle cell apoptosis was detected in arteries with dead cells replaced primarily by collagen. Despite the catastrophic effects on vascular smooth muscle, the deleted visceral smooth muscle remained viable with the exception of a short portion of the colon, indicating that vascular but not visceral smooth muscle is particularly sensitive to changes in β1-integrin.
Conclusions—This study reveals an essential function of β1-integrin in the maintenance of vasomotor control and highlights a critical role for β1-integrin in vascular, but not visceral, smooth muscle survival.
- cell adhesion
- extracellular matrix
- vascular diseases
- vascular fibrosis
- vascular occlusion
- vascular resistance
Vascular smooth muscle cells (vSMCs) provide critical support to the cardiovascular system through their ability to sense and respond to changes in transmural pressure.1,2 This function requires intimate association and interaction of SMCs with extracellular matrix (ECM) proteins. In fact, deletion or mutations in some of these proteins can lead to vascular dysfunction and aneurysms.3–8 The integrin family of ECM receptors resides at the interface between SMCs and the ECM; thus, it is anticipated that these molecules, along with the ECM, partake in the maintenance of vascular homeostasis. Although much has been clarified about the contribution of integrins during vascular development, the individual (or combined) role of these proteins in adult vascular homeostasis in vivo is not well understood.1
β1-Integrin is a particularly important member of the integrin family of heterodimeric receptors because it can pair with each of 10 different integrin α-subunits in vSMCs.1,9 In development, deletion of β1-integrin from smooth muscle results in aneurysms at the branch points of the aortic arch and it alters adhesion of isolated SMCs in vitro.4,5 In addition to its role in the assembly of the vascular wall, β1-integrin is thought to contribute to vasoregulation, serving as a mechanosensor in adult smooth muscle. For example, in isolated resistance arterioles, activation of integrins by ligand peptides can regulate Ca2+ channels on the plasma membrane triggering SMC contraction and vasoconstriction.10–12
The contribution of β1-integrin in the regulation of Ca2+ channels represents a potential role of this protein in vascular wall physiology. A second function for β1-integrin is to provide mechanical stability through its ability to anchor several elements of the cytoskeleton providing critical support (tensegrity) to SMCs.13,14 Integrins, together with other ECM receptors, are able to transmit forces experienced by the external environment to the cytoskeleton.15 Within the cell, the combination of stiff microtubules and the more flexible microfilaments and intermediate filaments provides increasing resistance to increased force,14 offering instantaneous responses to changes in stress. Integrins are also known to provide biochemical signals that regulate adhesion, migration, and survival. It is intriguing that these respective models are often described to the exclusion of the other. Instead, both the active signaling and the biomechanical tensegrity are likely to work in tandem to provide vasomotor control, particularly in the mature vascular wall. To further explore the biological contribution of β1-integrin in vascular structure and function, it is essential to evaluate these properties in mature and physiologically reactive vessels.
In the present study, we tested the hypothesis that β1-integrin is required to maintain the structure and function of vSMCs in an adult mammalian system. For this purpose, a β1-integrin exon 3 floxed mouse was crossed to the tamoxifen-inducible sm22αCre-ERT2 mouse.16,17 Mice were allowed to reach 5 weeks of age at which point β1-integrin deletion was induced using a 14-day tamoxifen regimen. The consequences of this deletion varied in severity according to vessel diameter but eventually resulted in apoptosis of vSMCs. Compared with controls, systemic arteries exhibited extensive loss of vSMCs with impaired force production and increased synthesis of ECM. In arterioles supplying the cremaster muscle, constriction to norepinephrine and dilation to acetylcholine (Ach) were muted. These findings demonstrate an essential role for β1-integrin in vSMC survival in conduit arteries and in the regulation of tissue blood flow in the microcirculation.
Materials and Methods
Materials and Methods are available in the online-only Supplement.
The β1-integrin promoter is constitutively active in adult smooth muscle. In adult vessels, both endothelial and SMCs express high levels of β1-integrin protein (Figure 1A). A β1-integrin reporter mice further confirmed that this gene is highly active in adult vessels. These reporter mice are double transgenic animals expressing a Cre transgene (sm22αCre or vascular endothelial-cadherin) and flox β1-integrin (β1fl) that when deleted enables lacZ gene expression in the cell-specific cell type that expresses cre recombinase (Figure I in the online-only Data Supplement). Examination of β1-integrin promoter activity in the reporter mice using β-galactosidase (β-gal) activity in embryonic and mature smβ1fl/wt mice indicated high promoter activity in smooth muscle of the dorsal aorta during development and in the adult (Figure 1B and 1C). The same analysis performed on mice expressing the endothelial-specific vascular endothelial-cadherin-Cre showed only sporadic β-gal–positive cells (Figure 1D). Both endothelial cells and smooth muscle maintain some level of β1-integrin protein; however, SMCs seem to require continuous promoter activity in the majority of cells. The persistence of β1-integrin promoter activity into maturity coupled with the continued presence of the protein in the adult led us to further inquire about the biological function of β1-integrin in mature smooth muscle using an inducible loss of function approach.
Conditional Deletion of β1-Integrin in Smooth Muscle
Conditional deletion of β1-integrin was achieved by crossing a β1e3/e3 floxed mouse to the sm22α-CreERT2 (inducible smooth muscle Cre [ism]) mouse model.16,17 To delete β1-integrin, a 2-week induction regimen was initiated at 5 weeks of age on ismβ1e3/e3 and β1e3/e3 controls. Polymerase chain reaction for both the intact allele (e3int) and the excised allele (e3ex) was performed on dorsal aortas from β1e3/e3 and ismβ1e3/e3 mice injected for 2 weeks and aged for an additional 2 weeks. Polymerase chain reaction confirmed recombination in the aorta of ismβ1e3/e3 mice (Figure 1E). Western blot analysis and immunofluorescence staining for β1-integrin verified the decrease in protein levels (Figure 1E and 1F). Note that cells in the intima and adventitia continue to express β1-integrin (Figure 1F) offering an explanation for the residual levels of protein noted in the Western.
To assess recombination efficiency, we crossed the sm22α-CreERT2 to a Rosa26R (R26R) transgenic mouse model. Cre-mediated recombination in the ismR26R offspring showed increasing β-gal–positive SMCs with increasing doses of tamoxifen (5–14) with minimal differences between 14 and 28 doses (Figure IIa in the online-only Data Supplement).18 vSMCs in vessels of the abdominal muscle, dorsal aorta, heart, and lung were examined at the 14-dose time point to determine the degree of recombination occurring in the ismβ1e3/e3 mice (Figure IIb in the online-only Data Supplement). Using this double transgenic model, recombination was also observed in the visceral smooth muscle of the lung, bladder, and esophagus. No β-gal–positive cells were identified in cardiac or skeletal muscle when animals were exposed to tamoxifen >4 weeks of age (Figure IIb in the online-only Data Supplement). The status of β1-integrin protein after 14 doses of tamoxifen (once per day) was evaluated in bladder, dorsal aorta, and small and large intestine by both Western blot and immunofluorescence (Figure III in the online-only Data Supplement). These findings demonstrated the specificity of sm22alpha-CreErT2 and corroborated previous results on the efficiency of this transgenic line.
On verification of the model and deletion approach, we proceeded to investigate the effect of β1-integrin inactivation in mature smooth muscle. For these experiments, young adult ismβ1e3/e3 and β1e3/e3 mice were treated with tamoxifen for 2 weeks and observed. Mice from the ismβ1e3/e3 group, but not from the control cohort, die between 9 and 15 weeks of age, which corresponds to 4 to 10 weeks after the initial tamoxifen treatment (Figure 1G). These findings demonstrated an essential requirement for β1-integrin in the SMC compartment and highlighted its critical role in survival.
β1 Deletion Mutes Contractile Vascular Response
Previous in vitro experiments identified the contribution of β1-integrin in vasoregulation. Specifically, peptides associated with β1-integrin ligands were shown to be vasoactive and inhibition of this receptor changes vascular reactivity.10–12 To determine the effects of β1-integrin deletion on vascular function, we studied the contractile properties of isolated vascular rings and the reactivity of arterioles controlling blood flow in the cremaster muscle of anesthetized mice. These functional studies were performed 6 weeks after the first tamoxifen injection.
Responses to norepinephrine (NE, 3×10−5 mol/L) and to 80 mmol/L extracellular [K+] (K80) were significantly reduced (P<0.05) for ismβ1e3/e3 versus β1e3/e3 in 3 of the 4 arteries studied (Figure 2A). Although maximal active tension was maintained in the aorta of ismβ1e3/e3, the superior mesenteric artery (SMA) was 61%, the SMA branch was 11%, and the femoral artery was 1% of values recorded for β1e3/e3 controls. A similar pattern was observed for NE-induced contractions (Figure 2A, right). With no significant difference for aorta, peak active tensions for SMA and SMA branch of ismβ1e3/e3 were 29% and 7% of responses in β1e3/e3, respectively, whereas responses of ismβ1e3/e3 femoral artery were abolished.
A trend similar to the one observed in vessel ring assays was also noted in cremaster arteriolar preparations. These showed loss of vascular responses to NE in ismβ1e3/e3 (Figure 2A and 2B). The physiological responses of the SMA at 6 weeks post-tamoxifen treatment can be compared with the histological status of the same artery at the identical time point (Figure IVc in the online-only Data Supplement). At this postdeletion time, the mesenteric artery shows significant loss of smooth muscle, identified in red by the trichrome staining, and progressive fibrosis in green (Figure IVc in the online-only Data Supplement). Such loss of SMCs is consistent with impaired response to vasoconstrictors. In contrast to the mesenteric artery, the aorta displayed no functional alterations at 6 weeks post treatment (Figure 2A) and histological examination revealed only minimal loss of SMCs in the layer near to the adventitia (Figure IVd in the online-only Data Supplement). Overall, the findings indicate that deletion of β1-integrin has more devastating effects in arteries of intermediate than in those of larger size like the aorta.
Endothelial cell–dependent vasodilation to ACh and vSMC-dependent vasoconstriction to NE was examined in cremaster arterioles in vivo (Figure 2B). First-order (1A), second-order, (2A) and third-order (3A) arterioles were examined under resting conditions and in the presence of cumulative concentrations of ACh and NE. Although baseline diameter in 1A arterioles was significantly smaller in ismβ1e3/e3 versus β1e3/e3 (P<0.05), 2A and 3A arterioles were not different between groups (Table). During superfusion with sodium nitroprusside and ACh, the maximum diameters of 1A, 2A, and 3A were each significantly (P<0.05) smaller in ismβ1e3/e3 versus β1e3/e3 (Table). Spontaneous vasomotor (ie, myogenic) tone was greater in 2A and 3A than in 1A for both β1e3/e3 and ismβ1e3/e3 mice, but was less in 2A and 3A of ismβ1e3/e3 versus β1e3/e3 (P<0.01; Table I in the online-only Data Supplement). Across branch orders, ACh had no effect on the diameter of arterioles in ismβ1e3/e3 mice (Figure 2B, top 3 panels). In contrast, arteriolar diameters increased with progressively higher concentrations of ACh in β1e3/e3 and were significantly greater (P<0.05) than ismβ1e3/e3 at the highest concentrations (Figure 2B, top 3 panels). For 1A in ismβ1e3/e3, a cumulative increase in NE concentration had no significant effect on diameter through 10−5 mol/L. For 1A in β1e3/e3, NE was not studied above 3×10−7 mol/L because of complete vasoconstriction of 3A. In 2A and 3A of β1e3/e3, vasoconstriction increased with cumulative addition of NE and 3A closure occurred with ≤10−6 mol/L (Figure 2B, middle). For 2A and 3A of ismβ1e3/e3, constriction to NE seemed to be attenuated versus β1e3/e3 (Figure 2B, middle) but there were no statistically significant differences between respective groups. On closer examination, distinct response profiles emerged for 2A and 3A in the ismβ1e3/e3 group. In half of these mice, there was no vasomotor activity in response to NE, whereas responses in the other half were similar to β1e3/e3 (Figure 2B, lower 3 panels). This difference between ismβ1e3/e3 responders and nonresponders was significantly different. Together these data indicate that deletion of β1-integrin from vSMCs diminishes both contraction and relaxation capacities.
Progressive Degeneration of the Tunica Media
The lack of vasomotor responses to ACh and NE indicated significant smooth muscle dysfunction. To assess the cellular changes associated with deletion of β1-integrin, coronary vessels from ismβ1e3/e3 and β1e3/e3 controls were evaluated at 5 days, 2 weeks, 3 weeks, 6 weeks, and 10 weeks from the first tamoxifen injection (Figure 3A). At 5 days, the coronary arteries of ismβ1e3/e3 resembled controls. At 2 and 3 weeks, the vSMCs of the ismβ1e3/e3 appeared less compact. By 6 weeks the coronary arteries had significantly lost vSMC coverage, which was even more pronounced at the 10-week time point. Although β1-integrin reduction was apparent by Western blot analysis and microarray as early as 2 weeks after treatment in mesenteric arteries (Figure IVa and IVb in the online-only Data Supplement), histological deterioration of the vSMC layer was only evident at 6 weeks. From 6 weeks progressive medial degeneration similar to that seen in the coronary arteries was noted in the mesenteric arteries (Figure IVc in the online-only Data Supplement). Matrix proteins occupied the space vacated by smooth muscle. Surprisingly, this loss of smooth muscle was not associated with an inflammatory response.
Recent reports indicate that a stem cell population resides within the adventitia. It has been proposed that these cells may contribute to vascular wall repair19,20 and give rise to SMCs. To assess potential stem cell investment during the degeneration of the media layer, ismβ1e3/e3 animals were crossed to animals carrying the R26R transgene. As the cre recombinase does not mark adventitial cells, if adventitial stem cells were able to integrate into the tunica media, those cells would be negative for β-gal. After the 2-week injection regimen, animals were aged an additional 4 weeks and the tissue was stained for β-gal (Figure V in the online-only Data Supplement). These representative sections indicate that β-gal–positive cells deleted for β1-integrin were not replaced by β-gal–negative stem cells after tamoxifen injections ended. This failure to rescue vSMCs was further corroborated by the progressive degeneration and fibrosis of the tunica media (Figure 3A and Figure IVc in the online-only Data Supplement).
The coronary vasculature provided insight on the effects of β1-integrin deletion; however, arteries supplying the heart may be subjected to physical stress not experienced by other vascular beds. To assess the systemic consequence of β1-integrin deletion in vSMCs, additional vascular networks and other SMC populations were evaluated. At 6 weeks, the dorsal aorta showed signs of tunica media degeneration, although the effect was not as significant as that observed in medium-sized arteries (Figure 3B and Figure IVd in the online-only Data Supplement). The mesenteric and hepatic arteries of the liver exhibited a marked degradation of the tunica media (Figure 3B). Further, all vascular beds examined showed some occluded vessels (particularly arterioles), especially the liver (Figure VIa and VIb in the online-only Data Supplement). Although we anticipated the coronary vasculature to be affected by β1-integrin deletion, we found that the effect was systemic and it was characterized by progressive vascular fibrosis with concurrent loss of SMCs (Figure 3A and 3B).
As endothelium-dependent dilation in arterioles was consistently impaired by β1-integrin deletion (Figure 2B), we examined the physical integrity of the endothelium of vessels with mutant SMCs. Immunofluorescence of α-smooth muscle actin (green) and platelet endothelial cell adhesion molecule (red) illustrates that deletion of β1-integrin clearly affects the tunica media, whereas the endothelium remains intact (Figure 3C).
To gain a better understanding of β1-integrin in SMCs, in general, we also examined the condition of visceral smooth in several organs. Analysis of the ismR26R intestine showed that cre recombinase was fairly penetrant, as indicated by β-gal–positive cells in the small and large intestine (Figure VIIb in the online-only Data Supplement). Surprisingly, histology of the small intestine showed no difference between ismβ1e3/e3 and β1e3/e3 controls, despite significant decreases in β1-integrin protein (Figures IIIa, IIIb, and VIIc in the online-only Data Supplement). The finding was also true for most of the colon, with the exception of a small region at the junction with the cecum (region 4 in Figure VIIc in the online-only Data Supplement). In the latter region, SMC loss and fibrosis were evident and similar to the effect noted in the vasculature. Visceral SMCs in the bladder and lung (not shown) between knockout and control animals were morphologically identical (Figure VIIa in the online-only Data Supplement). We were intrigued by the different outcomes of β1-integrin deletion in vascular versus visceral SMC populations. To gain insight into this difference, we evaluated levels of β1-integrin, β3-integrin, β5-integrin, CD36, galactosidase β1, ribosomal protein SA, and serum response factor by Western blot in the bladder, dorsal aorta, mesenteric arteries, small intestine, and large intestine of 2 wild-type mice (Figure VIII in the online-only Data Supplement). The prediction was that differential integrin expression patterns (or other receptors) could explain such a difference. We found that levels of β1-integrin were highest in bladder and similar between vascular and intestinal smooth muscle. A similar pattern emerged for β3 and β5 integrins. Furthermore, other receptors (CD36, galactosidase β1) were more specific to the vascular smooth muscle. These findings are in accordance with recent work demonstrating that vSMCs and visceral SMCs exhibit unique expression profiles21 and such differences may, in turn, contribute to differences in vulnerabilities to loss of β1-integrin found here. Remarkably, despite effective deletion of β1-integrin in visceral smooth muscle, the relative lack of consequences is in striking contrast to the devastating effect of β1-integrin deletion in vascular smooth muscle.
ECM Expression Is Increased in the Vasculature
Histological evaluation indicated that SMC loss in ismβ1e3/e3 vessels was associated with a corresponding increase in ECM. Given the large spectrum of ECM molecules secreted by SMCs, several of which are ligands for β1-integrin heterodimers, we performed microarray analysis and obtained information on ECM and other transcriptional changes triggered by loss of β1-integrin. For these experiments, we used RNA isolated from mesenteric arteries at the 2-week time point. This time was selected because the cells were beginning to show stress (Figure 3A) but had yet to undergo apoptosis. The microarray data indicate that transcripts for most ECM molecules were increased (Figure 4A). To further validate the findings and gain information on location, we performed immunofluorescence for fibrillar collagen, fibronectin, elastin, and laminin. The analysis was done in n=3 to 5 independent mice per group (β1e3/e3 and ismβ1e3/e3); representative images are shown in Figure 4B–4E. Sections of both coronary vessels and the dorsal aorta were examined by confocal microscopy (Figure 4B–4E and Figure IXa–IXd in the online-only Data Supplement). In the tunica media, fibrillar collagens (I, III, and IV) together with laminin replaced the SMCs in both the coronary vasculature and the dorsal aorta (Figure 4B and 4E). Although fibronectin was the predominant ECM within the adventitia of coronary vessels and dorsal aorta, the protein was not found to populate the media on deletion of β1-integrin (Figure 4C). There was little difference in elastin staining between wild-type and deleted vessels (Figure 4D), despite the changes in elastin transcripts. Changes in ECM production in the media are often associated with dedifferentiation of vSMCs (synthetic versus secretory phenotypes). Nevertheless, the microarray data did not indicate statistically significant changes in expression of myosin or calponin. Immunostaining of smooth muscle myosin, α-smooth muscle actin, vimentin, and α-tubulin were consistent with the microarray findings of a relatively intact cytoskeleton in a differentiated SMCs (Figure X and Table I in the online-only Data Supplement). Overall, these findings reveal differential deposition of matrix proteins with a preponderance of fibrillar collagens in the tunica media of ismβ1e3/e3. In turn, the increased deposition of collagen within the vascular wall is likely to contribute to the altered functional response of vessels in mutant mice (Figure 2).
vSMCs Undergo Apoptosis
In addition to increased ECM, the coronary vessels of ismβ1e3/e3 mice clearly depicted loss of SMCs. As β1-integrin has been shown to function in cell survival in vitro, we evaluated SMC loss in ismβ1e3/e3 mice. A systematic analysis of SMC content was applied in ismβ1e3/e3 and β1e3/e3 mice using lumen area to classify vessels according to size. As anticipated, we found loss of smooth muscle in ismβ1e3/e3 mice; however, at the time point examined (6 weeks post deletion), only vessels from ismβ1e3/e3 mice with a lumen area >1000 μm2 were found to have a significant reduction in SMC coverage compared with β1e3/e3 (P>0.05; Figure 5A and 5B). These data indicate that SMCs in medium-sized arteries are more sensitive to loss of β1-integrin and are consistent with the functional data from arterial rings (Figure 2A). To determine whether this loss in smooth muscle was because of apoptosis, heart sections were stained for cleaved caspase 3 (red) with α-smooth muscle actin (green) marking the tunica media. No cleaved caspase positive clusters were identified in the β1e3/e3 control (228 SMCs counted across 3 animals). In contrast, >40 positive clusters were seen among the ismβ1e3/e3 vessels examined (260 SMCs across 3 animals). Images of coronary arteries from 2, 6, and 8 weeks after the first injection further confirm that the progressive loss of SMCs attributes to apoptosis (Figure 5C). The prevalence of apoptosis within the tunica media indicates that β1-integrin is in fact critical to SMC survival. This finding is in sharp contrast to the lack of apoptosis in visceral smooth muscle, with the unique exception of a small region in the colon (Figure VIIc in the online-only Data Supplement).
Compensatory Response by Visceral But Not Vascular Smooth Muscle
The striking differences between the phenotype of vascular and visceral smooth muscle let us to further consider potential compensatory differences in integrin expression. As discussed previously, β1, β3, and β5 integrins are expressed by both vSMCs and visceral SMCs at similar proportions. Meaning, SMCs of both types have high levels of β1 and β3, followed by lower levels of β5 (Figure VIII in the online-only Data Supplement). On loss of β1-integrin in vSMCs, we observed a slight trend, but no significant increase in β3-integrin (Figure 6A and 6B). Conversely, visceral SMCs exhibited a 3-fold increase in β3 on the loss of β1-integrin (Figure 6A and 6B). No changes were detected in the level of expression of β5 upon β1-integrin deletion in either visceral or vascular cells (Figure 6C).
The findings indicate a potential mechanism for the lack of phenotype in visceral SMCs namely functional compensation by β3-integrin. Interestingly, vSMCs were not able to mount such a response on the loss of equivalent levels of β1-integrin. The reason(s) for this difference is unclear and highlights the strict dependency of vSMCs for this receptor.
Previous reports have shown an essential role for β1-integrin in the assembly of the vascular wall during development.4,5 Here, we examined the contribution of β1-integrin in adult SMCs by selectively inactivating this gene in young adult mice. We demonstrate that deletion of β1-integrin results in a reduced contractile response of arterial rings, impaired constriction and dilation of arterioles in vivo, progressive systemic fibrosis, and eventual death of SMCs.
Animals with induced deletion for β1-integrin in SMCs remained physically active for several weeks, but deletion eventually resulted in their demise. Although it is difficult to determine the exact cause of death, the ismβ1e3/e3 surviving for 10 weeks post-tamoxifen treatment showed several fibrotic vessels with partially or fully occluded lumens. In addition, mice became bloated from intestinal impaction. This is likely because of the fibrosis of a small area of the colon that is sensitive to β1-integrin deletion and undergoes apoptosis (Figure VIIc in the online-only Data Supplement region 4). At 10 weeks, ismβ1e3/e3 mice seemed to have less adipose tissue and become moribund at approximately the same time. We did not see any obvious signs of aneurysms or dissections in any of the animals examined.
Although we noted a clear effect of β1-integrin deletion in vascular tone of arterioles in vivo, phenotypes varied according to vessel caliber. Remarkably, contractile responses of the aorta showed little consequence to the loss of β1-integrin. This may be because of the high elastic content of the aorta, storing energy during systole, and releasing it during diastole to facilitate driving blood flow to the periphery.22 It is also possible that this vessel experiences less recombination (Figure IIa and IIb in the online-only Data Supplement). In contrast, medium size arteries rely mostly on the contractile function of smooth muscle, which showed extensive SMC apoptosis and a significantly depressed response in ismβ1e3/e3 animals (Figures 2A and 5).
The absence of constriction to NE in ismβ1e3/e3 mice is consistent between the 1A arterioles and the vascular rings from the SMA branch and femoral artery (Figure 2A and 2B) of these mice when compared with β1e3/e3. That such differences were observed to K80 and NE of arterial rings points to a generalized functional deficit in the ability of SMCs to contract in response to either membrane depolarization or adrenoreceptor activation, respectively. The loss of contraction to NE in 2A and 3A that was manifested in half of the ismβ1e3/e3 group (Figure 2B) suggests that the penetrance of this functional deficit in arteriolar SMCs was ≈50% at the 6-week time point.
Our findings in arterioles of the cremaster muscle illustrate that endothelium-dependent vasodilation to ACh was impaired, along with vasoconstriction to NE, despite the presence of an intact endothelium. In vascular rings, contractile responses to NE and depolarizing K+ were dramatically compromised in muscular arteries. In turn, impaired dilation and lack of constriction in arterioles would underscore poor regulation of blood pressure and tissue blood flow, whereas impaired constriction of muscular arteries would contribute further to such an effect. The consistency across vascular branch orders implies that β1-integrin is important for vasomotor control.
Across branch orders, arterioles of ismβ1e3/e3 have diminished endothelium-dependent vasodilation. When sodium nitroprusside (an NO donor) was added together with ACh to obtain maximal diameters, dilation of 2A and 3A (Table) indicates that vSMCs of these arterioles in ismβ1e3/e3 can still relax to exogenous NO, albeit to a lesser extent than β1e3/e3. With similar resting diameters for 2A and 3A between groups, the reduced tone (ie, smaller maximal diameters) in 2A and 3A of ismβ1e3/e3 reflects the inability of SMCs to relax and remodel to smaller diameters. Collectively, the reduction in maximal diameter across branch orders is consistent with arteriolar remodeling after β1-integrin deletion. That deficits in arteriolar function, including impaired vasodilation, are caused by defective SMCs rather than impaired endothelium is consistent with the lack of inflammatory response seen throughout the vasculature, with intact endothelial cells maintaining this protective role, despite SMC apoptosis.
In addition to impaired functional responses from ismβ1e3/e3 vessels, histological evidence showed a progressive degradation of the tunica media. This deterioration continued after tamoxifen injections had ceased. The design of this experiment also enabled us to examine potential repopulation of the tunica media through adventitial stem cells. Sca1+ (vascular stem cells) have been observed in the adventitia of the aortic root and express some smooth muscle differentiation markers.20 In our early studies, we applied a 28-day injection regimen and found significant smooth muscle degeneration continuing until the animals were found moribund at ≈8 weeks from the first tamoxifen injection (data not shown). The 2-week injection protocol was later found to be sufficient to induce significant deletion. The ismβ1e3/e3 animals survived for several weeks after the final injection. From the last injection until death the medial layer continued to degenerate with no visible gain of potential cells from the adventitia. β-gal staining of ismR26R mice was compared with ismβ1e3/e3; R26R mice indicate that unmarked cells of the adventitia do not repopulate the vascular media. In fact, there was invasion of neither adventitial cells nor any inflammatory cells into the media. Data from Clarke et al23 also suggest that vSMC apoptosis does not stimulate a compensatory or replacement mechanism. It is possible that the lack of recovery may reflect the timing of β1-integrin deletion. The Sca1+ progenitor population is reduced in maturation, but these data indicate, that at least within this time frame, adventitial stem cells are not active in mature vessels and do not contribute toward the healing of an impaired vascular wall.20 Alternatively, the fibrotic condition may disrupt the stem cell differentiation seen in apolipoprotein E–deficient mice.24
Rather than an inflammatory response, the loss of β1-integrin and cell death resulted in the production of ECM as the primary response to progressive degeneration of the tunica media. In fact, an interesting aspect of this phenotype is the transition of vSMCs to a secretory state, but in the absence of drastic dedifferentiation (as noted in vitro) and before initiation of apoptosis. The present data would indicate that lack of tension triggered by defective vSMC binding to the ECM results in significant synthesis and secretion of matrix proteins, perhaps in an attempt to regain the previous tensile state (Figure 4). The increase in matrix might indeed represent the mechanism described by Tomasek et al25 where myofibroblasts secrete excess matrix until a set tension is achieved and an attempt of the defective smooth muscle to regain attachment with its adjacent matrix after losing tension from vSMC loss. It should be stressed, however, that the staining reveals deposited ECM and not necessarily measures secreted matrix proteins. The ability of transglutaminase to cross-link matrix proteins requires β1-integrin, thus it is possible that these deleted cells might also secrete fibronectin but are unable to incorporate this protein in the remodeled matrix.
Developmental deletion of β1-integrin from smooth muscle is not associated with apoptosis, although the time frame of development deletion versus induced deletion in adult is slightly different and perhaps the reason for the absence of apoptosis.4,5 Nonetheless, β1-integrin inactivation in the development also resulted in abnormal ECM assembly and this information can provide important points of comparison with the adult deletion. During development, excess ECM was deposited in areas of high stress. In the adult, ECM deposition correlates with SMC loss. Although β1-integrin was inactivated in all SMC populations, SMC death occurred mostly in large- and medium-sized arteries with the exception of the aorta, which may be buffered from stress by its elastin content in addition to lower recombination efficiency. The smallest vessels neither lost smooth muscle nor accumulated excess ECM. In the vascular hierarchy, smaller vessels maintain a lower pressure than larger vessels.26 The data suggest there may be a correlation between the higher vascular pressure of medium arteries and cell death after β1-integrin deletion. This interpretation is further corroborated by the lack of apoptosis in the small intestine and bladder where pressure is generally at 20 mm Hg, whereas the colon sees spikes >40 mm Hg.27,28
In adult vascular tissues, the ultimate consequence of β1-integrin loss is apoptosis, which occurs in the absence of a robust inflammatory response. The lack of inflammation correlates with models of diphtheria toxin in which induced SMC apoptosis did not result in an inflammatory response.23,29 Curiously, diphtheria toxin–induced apoptosis did not produce a fibrotic response. By comparison, a mouse carrying a mutated copy of lamin A showed both SMC death and fibrosis.30 These differences between models may suggest that fibrosis after SMC death results from death in the context of an altered/stressed population of SMCs (slow death), but that rapid apoptotic events do not induce ECM production.
In conclusion, this work illustrates the critical role β1-integrin in SMC survival and function in vivo. By considering the consequences of deletion in vivo we gain valuable insight into potential mechanisms of disease, possible therapies, and their side effects. The ismβ1e3/e3 phenotype resembles the medial degeneration and vascular fibrosis seen in Raynaud syndrome and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, yet lacks an inflammatory component. There are also similarities in the vascular phenotype between a lamin A mutant mouse model of progeria and the smooth muscle β1-integrin knockout.30 The model presented here provides a unique opportunity to distinguish the molecular mechanism driving the inflammatory response versus the mechanism of fibrosis. Further, these findings may offer insight into the side effects of anti-integrin therapies, which are being used for the treatment of Crohn disease and multiple sclerosis. With the knowledge that β1-integrin contributes to vasoregulation and cell survival, perhaps more specific therapies can be developed and adverse effects avoided based on our understanding of β1-integrin in adult vSMCs.
We thank the contribution of the Tissue Procurement Core Laboratory Shared Resource for their support in histological processing of our samples. Experiments other than those in Figure 2 were executed by M.L. Iruela-Arispe, K.A. Turlo, and J. Scapa; experimental design and interpretation by M.L. Iruela-Arispe and K.A. Turlo; and article by K.A. Turlo and M.L Iruela-Arispe. Experiments on vessel rings were performed by A.W. Jones in the laboratory of R.J. Korthuis. Experiments on cremaster muscle arterioles were performed by P. Bagher in the laboratory of S.S. Segal. For the experiments presented in Figure 2, all investigators contributed to experimental design and data interpretation. A.W. Jones and P. Bagher prepared the first draft of these results. S.S. Segal prepared the documents required for shipping and care of these animals for the University of Missouri and edited the final submission of this report. Transgenic sm22αCre-ERT2 mouse was obtained from R. Feil.
Sources of Funding
This research was supported by National Institutes of Health grants RO1-HL085618 (M.L. Iruela-Arispe), R37-HL041026 and RO1-HL086483 (S.S. Segal), F32-HL097463 (P. Bagher), and RO1-HL095486 (R.J. Korthuis).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300648/-/DC1.
- Received October 18, 2012.
- Accepted June 16, 2013.
- © 2013 American Heart Association, Inc.
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This work defines the role of β1-integrin in mature smooth muscle cells in vivo. Using a tamoxifen-inducible sm22αCre recombinase to delete the itgb1 gene in a manner that bypasses developmental stages, we show that loss of β1-integrin in adult smooth muscle initially impairs vascular contractility/dilatory responses and eventually results in cell death. After deletion of β1-integrin, vessels become fibrotic with investment of collagen and other extracellular matrix molecules but in the absence of an inflammatory response. Finally, this study highlights differences between vascular and visceral smooth muscle cells to loss of β1-integrin, indicating an important divergence in these 2 cell populations. We also highlight the relevance of in vivo studies to definitively determine the unique function integrins within the context of a given organ.