Integrin β1 Subunit Controls Mural Cell Adhesion, Spreading, and Blood Vessel Wall Stability

Animal Models

Mice carrying a loxP-flanked Itgb1 gene (Itgb1^lox/lox) and Pdgfrb-Cre have been reported previously.^16,17 For the generation of mutants, Pdgfrb-Cre Itgb1^lox/+ double heterozygotes were bred to Itgb1^lox/lox mice in a mixed 129×C57BL/6 genetic background. Cre-negative littermates were used as controls. H-2KbtsA58 immorto¹⁸ or Rosa26–enhanced yellow fluorescent protein (EYFP) Cre reporter transgenes¹⁹ were introduced for the isolation of immortalized VSMCs or flow cytometric experiments, respectively. All animal experiments complied with the relevant laws and were approved by the Cancer Research UK Animal Ethics Committee.

Analysis of Tissues

For immunofluorescence on sections, tissue samples were fixed overnight in 4% paraformaldehyde at 4°C and embedded in paraffin for sectioning. Microtome sections were blocked with 0.1% BSA and 1.5% goat serum in PBS (30 minutes) before overnight incubation with primary antibody diluted in blocking solution. Primary antibodies were rat monoclonal anti–integrin β1 (Chemicon, 1:100), rabbit polyclonal anti-fibronectin (Sigma, 1:200), anti–collagen IV (Chemicon, 1:200), anti–phospho-histone H3 (Upstate, 1:100), and goat polyclonal anti–smoothelin B (Santa Cruz Biotechnology, 1:100). After washing, samples were incubated in secondary antibody (anti-rabbit Alexa Fluor-488 or Fluor-546, anti-rat Alexa Fluor-488, Molecular Probes, 1:500) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, 1:1000). Fluorescence was visualized using a Leica DM IRBE light microscope.

For whole-mount staining, skin samples were fixed overnight in 4% paraformaldehyde, washed with PBS, blocked in 1% goat serum in PBS containing 0.1% Tween 20 (3 hours at room temperature), followed by overnight incubation with primary antibodies (diluted in blocking solution) at 4°C. Samples were washed 3 times (1 hour each) in PBS, incubated for 3 hours at room temperature with secondary antibodies diluted in blocking solution, and washed as before. Primary antibodies were rat anti-mouse platelet endothelial cell adhesion molecule-1 MEC13.3 (Pharmingen, 1:100), mouse anti-human α-smooth muscle actin (SMA) (Clone1A4, Sigma, 1:400), polyclonal rabbit anti-desmin (Abcam, 1:200), rat monoclonal anti-endomucin (gift from Dietmar Vestweber, Max-Planck-Institute for Molecular Biomedicine, Münster, Germany), and rabbit anti-fibronectin (Sigma, 1:400). A Zeiss LSM510 Meta was used for confocal microscopy. Sample analysis by electron microscopy has been described previously.¹⁷

Flow Cytometry

Mesenteric tissue from E17.5 or postnatal day (P) 2 Pdgfrb-Cre Itgb1^lox/lox Rosa26-EYFP or control mice was incubated for 2 hours at 37°C in 2 mL of PBS containing 400 U/mL collagenase (GIBCO). After the addition of 10 mL of medium (10% FCS in DMEM), cells were dispersed with a Pasteur pipette, sieved (pore size, 100μm), collected by centrifugation (1000 rpm, 5 minutes) and washed with 5 mL of medium. Cells were resuspended in 500 μL of DMEM containing 2% FCS, incubated with integrin β1 antibody for 45 minutes and anti-rat secondary antibody conjugated to Alexa Fluor-647 (Molecular Probes). Propidium iodide (Invitrogen) staining selected live cells, which were analyzed with a FACSCalibur system (BD Biosciences).

Smooth Muscle Cell Isolation, Culture, and Analysis

The isolation of SMCs from adult aortas, as well as the culture, verification, transfection, and staining of these cells; the analysis of fibronectin fibrillogenesis; and video microscopic and automatic cell shape analysis are described in the online data supplement, available at http://circres.ahajournals.org.

Targeting of Itgb1 in Mural Cells

Itgb1^Pdgfrb-Cre mutants, generated by breeding Pdgfrb-Cre transgenic mice¹⁷ into a background of animals carrying a loxP-flanked version of the Itgb1 gene (Itgb1^lox/lox),¹⁶ were obtained at E17.5 almost at the expected Mendelian ratio (18.8% instead of 25%) predicted for our breeding scheme (see Materials and Methods; Figure 1B). Even though these embryos were of normal size and appeared healthy, their skin contained visibly dilated blood vessels (Figure 1A). After birth, a gradually increasing fraction of mutants died and only 3 survivors were obtained beyond P10 (Figure 1B). Immunofluorescence with anti–integrin β1 antibodies on skin sections from P2 animals validated our genetic approach. Whereas the anti–integrin β1 signal labels both endothelial cells and VSMCs in control littermates, specific staining is only visible in the Itgb1^Pdgfrb-Cre endothelium but absent from α-SMA–positive cells (Figure 1C). Residual mural integrin β1 expression in mutant survivors at later postnatal stages suggests incomplete gene inactivation, and we therefore excluded these animals from our study. Flow cytometric analysis showed that integrin β1 is gradually depleted during development and that the majority of Pdgfrb-Cre–positive cells in postnatal mutants (74.2% at P1) has lost the protein (Figure 1D).

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Figure 1. Gene targeting of Itgb1 in mural cells. A, Phenotype of freshly isolated E17.5 embryos. The bottom images show higher magnifications of insets. Note dilated blood vessels in the Itgb1^Pdgfrb-Cre skin (right). B, Number of Itgb1^Pdgfrb-Cre mice (mutants) among the total of embryos/pups analyzed at indicated stages. Percentage (%) of mutant vs total mice relative to the expected ratio (25% for this breeding scheme) indicates lethality of Itgb1 mice. C, Double immunofluorescence with antibodies against integrin β1 (green) and α-SMA (red) on skin sections from P2 animals, confirming the absence of integrin β1 in VSMCs but not the endothelium of Itgb1^Pdgfrb-Cre mutants (right). Bottom images show individual channels of insets at higher magnification. Nuclei were stained with DAPI. Arrows indicate mutant or control VSMCs. D, Flow cytometric analyses of integrin β1 expression cells isolated from Pdgfrb-Cre Itgb1^lox/lox ROSA26-YFP Cre reporter mice at E17.5 or P1. Cells from ROSA26-YFP (without anti–integrin β1 antibody) and Pdgfrb-Cre ROSA26-YFP double transgenic embryos at E17.5 were used as controls, as indicated. Numbers in quadrants (purple boxes) indicate fractions of integrin β1–expressing (top) and YFP-positive (Cre-expressing) integrin β1–negative (bottom right quadrant) cell populations relative to total cells. Scale bars: 1 mm (A) and 20 μm (C).

Vascular Smooth Muscle Cell Defects in Itgb1 Mutants

Analysis of the Itgb1^Pdgfrb-Cre dermal vasculature by whole-mount immunofluorescence revealed the presence of vascular aneurysms, local distensions of blood vessels, affecting both arteries and veins already in embryos (Figure 2A and 2B). These aneurysms correspond with regions with poor VSMC coverage, suggesting that this phenotype is linked to mural cell defects. At P2, mutant VSMCs show a highly rounded, button-like morphology. Because of poor spreading, individual cells are often separated by gaps so that Itgb1^Pdgfrb-Cre arteries and veins lack continuous SMC coverage (Figure 2C and 2D). Staining with anti–α-SMA antibodies shows that the cytoskeleton of control VSMCs is aligned into a sheet of parallel fibers that surrounds the endothelium tightly. By contrast, Itgb1 mutant mural cells are highly disorganized and fail to align with neighboring cells. Moreover, α-SMA is no longer polarized to one surface of the VSMCs in the absence of integrin β1 (Figure 2E).

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Figure 2. VSMC defects in Itgb1 mutants. A through E, Confocal images of E17.5 (A and B) or P2 skins (C through E) after whole-mount immunostaining with antibodies detecting ECs (platelet endothelial cell adhesion molecule-1, green) and/or VSMCs (α-SMA, red). B and D, Higher-magnification images of insets in A and C, respectively. Arteries (a) and veins (v) are labeled. Arrows indicate aneurysm in A and B and rounded VSMCs in D and E. F, Electron micrographs of dermal blood vessels. Black arrows point at loosely associated Itgb1^Pdgfrb-Cre VSMCs. Endothelial (ec) and blood cells (bc) are labeled. Scale bars: 50 μm (A and C), 20 μm (E), and 2 μm (F).

On the ultrastructural level, mutant mural cells fail to associate with the subendothelial basement membrane, appear rounded and poorly spread, and are frequently located in some distance from the endothelium. As a consequence, Itgb1 vessel walls are not covered by a layer of tightly packed VSMCs and instead appear loosely organized (Figure 2F).

Integrin β1 Controls the Spreading of PCs

Because PCs play critical roles in the stabilization of capillary beds^2,3 and are targeted by the Pdgfrb-Cre transgene,¹⁷ we analyzed the morphology PCs in Itgb1 mutants. Whole-mount staining with antibodies directed against desmin, an intermediate filament protein and PC marker, allows the visualization of the PCs that cover the dermal vasculature with an extensive lattice of fine processes (Figure 3A). Desmin-positive PCs are also present in the Itgb1^Pdgfrb-Cre skin, and their number is even significantly increased compared with control littermates (Figure 3A and 3E). However, mutant cells lack the characteristic slender and stretched morphology of normal PCs, and their processes appear short (Figure 3B). Several lines of evidence suggest that loss of integrin β1 affects the interaction of PCs with ECs. In electron micrographs, mutant PCs have a round morphology, fail to wrap around dermal capillaries, and areas of endothelial-PC contact are small (Figure 3D). Whereas control PCs show no appreciable α-SMA immunofluorescence, mutant PCs are SMA-positive, similar to what has been reported previously for the poorly attached PCs covering the tumor vasculature.²⁰ Furthermore, distended capillary diameters in the Itgb1^Pdgfrb-Cre skin suggest that mutant PCs, despite their presence in greater numbers, are not supporting the endothelium sufficiently (Figure 3E). In contrast to other mutant models in which the loss or defective association of mural cells leads to edema formation and hemorrhaging,^17,21 we did not find extravasated red blood cells in Itgb1 mutants. The angiogenic growth of the vascular network, as judged by the size of the vascularized area (Figure IA in online data supplement), the number of blood vessel branch points, and endothelial proliferation (data not shown), was also not significantly altered when mural cells had lost integrin β1 expression.

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Figure 3. Defective morphology and association of Itgb1 PCs. A through C, Whole-mount staining of control and Itgb1^Pdgfrb-Cre dermal vasculature with anti-desmin (green), anti–α-SMA (red or blue), and/or anti-endomucin (red) antibodies as indicated. Arteries (a) and veins (v) are indicated in A. Desmin-positive PCs lack long cellular processes (arrows in B) and are more abundant (asterisks label cell bodies in B). C, Channel with anti–α-SMA signal of images shown in B. D, Electron micrographs of dermal capillaries. Itgb1^Pdgfrb-Cre mutants lack PC processes seen around the endothelium in controls (black arrows). PCs (pc), endothelial (ec), and blood cells (bc) are indicated. E, Quantitative analysis showing larger capillary diameters (5.0±0.4 vs 6.7±0.7 μm; *P=0.011 by 2-tailed t test assuming unequal variances) and increased PC numbers (48.5±6.2 vs 61.8±2.6 per 0.04 mm² area; *P=0.017 by 2-tailed t test assuming unequal variances) in P2 Itgb1^Pdgfrb-Cre head skin. Values are ±SD and based on 4 mutant and control samples, respectively. Scale bars: 50 μm (A), 20 μm (B), and 2 μm (D).

Integrin β1 Controls the Shape, Adhesion, and Motility of Cultured Mural Cells

To investigate the role of integrin β1 in cultured mural cells, we isolated aortic smooth cell cells from adult Itgb1^lox/lox homozygous mice, which also carried the H-2Kb-tsA58 immorto transgene, allowing the inducible expression of a temperature-sensitive SV40 T antigen.¹⁸ Transient expression of Cre recombinase in these cells yielded integrin β1–deficient (Itgb1^KO) SMCs. Deletion of the loxP-flanked region in the Itgb1 gene of these cells was verified by genotyping PCR (data not shown), and immunofluorescence confirmed the absence of integrin β1 protein (Figure 4A and 4B). Mock-transfected cells were used as controls (Figure 4A and 4B). Visualization of the focal adhesions by anti-paxillin antibody staining and of the actin cytoskeleton shows that loss of Itgb1 in cultured VSMCs phenocopies morphological changes observed in vivo. Itgb1^KO SMCs are round, poorly spread, and lack cellular protrusions seen in control cells (Figure 4A and 4C through 4E). Absence of integrin β1 expression does not prevent the formation of focal adhesions, even though they are short and disorganized in comparison with control cells (Figure 4A and 4B). As expected, reexpression of green fluorescent protein–tagged integrin β1 in Itgb1^KO SMCs restores their morphology, so that they resemble control cells (supplemental Figure II).

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Figure 4. Characterization of cultured integrin β1–deficient VSMCs. A, Anti–integrin β1 (red) and anti-paxillin immunofluorescence of control and integrin β1–deficient (Itgb1^KO) aortic SMCs, as indicated. B, Details of individual channels of insets in A. Arrows indicate integrin β1–positive focal adhesions (upper left). Note focal adhesion morphology is abnormal in Itgb1^KO cells (bottom right). C, Organization of the actin cytoskeleton (phalloidin, green) in control and mutant cells. Focal adhesions are labeled by anti-paxillin staining (red). Arrows indicate cortical actin in Itgb1^KO VSMCs. D, Example images of phalloidin-stained control (left) and Itgb1^KO (right) cells from automatic cell shape analysis (see Materials and Methods). Automatically identified and analyzed cells are in green and rejected cells are in orange. E, Histogram of shape factor data. Height of bars indicates cell numbers. Larger shape factor of integrin β1–deficient cells indicates decreased complexity of cell outlines, that is, more rounded cell shapes. Scale bars: 10 μm (A, C, and E).

Consistent with the binding affinities of β1-containing integrin heterodimers, attachment and spreading on collagen I and fibronectin matrix substrates is delayed in Itgb1^KO SMCs (Figure 5A and 5B). Similarly, cell motility and the persistence of migration are significantly reduced in the absence of integrin β1 (Figure 5C and 5D and supplemental Figure I).

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Figure 5. Regulation of cell adhesion and motility by integrin β1. A, Diagram showing the spreading of control and Itgb1^KO cells on collagen I or fibronectin substrates. Fraction of spread cells at indicated times were determined by video microscopy (see Materials and Methods). B, Still images of video microscopy showing areas containing spread (black arrowheads) and unspread (white arrowheads) control and mutant cells 90 minutes after plating. C, Diagrams showing tracks from control (n=16) and Itgb1^KO (n=13) cells with starting points shifted to the origin. Distances are indicated (in μm). Similar tracks were recorded in 2 further repeat experiments but are not shown in the figure. D, Graphic representation of migration speeds (μm/h) of control (left, n=16) and Itgb1^KO cells (right, n=13). Circles correspond to median speeds, boxes to 50%, and whiskers to 80% of values. Migration of mutant cells (14.0±1.4 μm/h) is significantly reduced compared with control cells (24.6±2.2 μm/h). ***P<0.001 by ANOVA.

ECM Protein Expression in Itgb1 Blood Vessels

Previous work has indicated that integrins are not only necessary for cell–matrix adhesion but, at least in some tissues, for the proper expression and deposition of ECM proteins.²² Evaluation of laminin, fibronectin, and collagen IV by immunofluorescence shows that all these matrix proteins are expressed in Itgb1^Pdgfrb-Cre dermal blood vessels (Figure 6A through 6C and supplemental Figure III). However, we noted that Itgb1 α-SMA–positive cells protrude through the matrix layer into the surrounding dermis and are no longer fully ensheathed by collagen IV, as is the case for control blood VSMCs (Figure 6A and 6B). Whereas fibronectin is still located within the basement membrane that separates the endothelium from the VSMC layer, its assembly into long parallel fibrils is defective in Itgb1^Pdgfrb-Cre mutants (Figure 6C and 6D). Confocal analysis reveals that mutant blood vessels lack long fibronectin fibrils and the ECM protein is instead predominantly accumulated in a circular fashion underneath VSMCs (Figure 6D). Similarly, cultured Itgb1^KO SMCs are only capable of limited fibronectin fibrillogenesis (Figure 6E).

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Figure 6. Matrix protein expression in Itgb1^Pdgfrb-Cre blood vessels. A through E, Immunofluorescence with antibodies directed against fibronectin or collagen IV (col IV) and α-SMA (red), as indicated. Sectioned P2 skins (A through C), confocal images of whole-mount samples (D) or immunostaining of cultured Itgb1^KO and control SMCs (E) are shown. B, Higher-magnification images of insets in A. Arrows point to rounded VSMCs protruding through the collagen IV layer. D, Long fibronectin fibers (arrows) in the proximity of α-SMA–positive VSMCs are present in control but not in Itgb1^Pdgfrb-Cre dermal blood vessels. E, Itgb1^KO cells display limited fibrillogenesis of plated fibronectin compared with control SMCs, which are capable of assembling long fibers (arrows). Nuclei in A through C and E were stained with DAPI (blue). Scale bars: 20 μm (A, C, and D) and 10 μm (E).

Proliferation and Differentiation of Itgb1 VSMCs

To gain a better understanding of the defects in mutant vessel walls, we evaluated whether the loss of integrin β1 has any effect on the survival, proliferation, or differentiation of mural cells. TUNEL staining shows no overlap between apoptotic (TUNEL-positive) nuclei and anti–α-SMA antibody signal in both control and mutant skin sections (data not shown), arguing against an essential role of integrin β1 in the protection of mural cells against cell death. By contrast, VSMC proliferation, assayed with antibodies against the mitotic marker phospho-histone H3, is increased in Itgb1^Pdgfrb-Cre mutants (Figure 7A through 7C). Thus, changes in the morphology of Itgb1 VSMCs are accompanied by upregulated proliferation reminiscent of the SMC behavior seen in response to vascular injury.

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Figure 7. Loss of integrin β1 affects VSMC proliferation and differentiation. A, B, D, and E, Immunofluorescence on P2 skin sections with antibodies against phosphorylated histone H3 (p-histone H3, green), smoothelin B (green), and α-SMA (red) as indicated. Nuclei were stained with DAPI (gray). Staining of red blood cells (*) is unspecific. B, The isolated green channel of mutant image in A. Arrows point at proliferating VSMCs in (A and B) and at control or Itgb1 VSMCs in E. C, Quantitation of p-histone H3–stained mutant and control blood vessel section. Bars represent proportion of control or Itgb1 vessels with a certain VSMC proliferation index (mitotic VSMCs/total VSMCs). Ninety-five percent of control sections contain no phosphorylated histone H3–stained VSMCs. E, Green channel (smoothelin B) of images shown in D. Scale bars: 20 μm (A through E).

The loss of late differentiation markers is another feature of the synthetic VSMC phenotype.^4,15 We therefore analyzed the expression of smoothelin B, an actin-binding protein that is a late VSMC differentiation marker linked to SMC contractility.²³ Whereas smoothelin B is abundantly present in α-SMA–positive cells of control vessels, such staining is missing in the Itgb1 dermal vasculature (Figure 7D and 7E). The expression of various transcriptional factors with established roles in the regulation of the smooth muscle differentiation program is also reduced and/or delayed in cultured integrin β1–deficient SMCs (supplemental Figure IV). All these findings together are consistent with a failure of VSMCs to acquire a fully differentiated and functional phenotype in the absence of integrin β1.

Regulation of Mural Cell Function by Integrin β1

Our findings demonstrate that integrin β1 is a critical regulator of mural cell morphology and function in vivo and in vitro. In the skin of Itgb1^Pdgfrb-Cre mutants, VSMCs are poorly spread, lack the normal alignment with neighboring cells, and support the vasculature insufficiently. This leads to poorly organized blood vessel walls and the formation of aneurysms. Similarly, mutant PCs, albeit present in increased numbers, fail to extend the long processes that are characteristic for this cell type and lack proper interactions with the endothelium. Ectopic expression of α-SMA by integrin β1–deficient PCs is highly reminiscent of PC defects in the deregulated vasculature of tumors²⁰ and most likely reflects changed gene expression in response to the loose association of these cells with the capillary network. The dilation of capillaries in the Itgb1^Pdgfrb-Cre skin is also consistent with insufficient support by mural cells. Even though we were unable to establish that these vascular defects were directly responsible for the lethality of the mutant mice, some form of cardiovascular insufficiency or the rupture of dilated and fragile blood vessels are the most likely cause of death. Embryonic and perinatal growth and development is accompanied by a significant increase in blood pressure,^24,25 and the severity of the mural cell defects suggests that the Itgb1^Pdgfrb-Cre mutant vasculature may be too weak to cope with these stronger hemodynamic forces.

Because many of the known molecular players controlling mural cell biology are linked to integrins, it is worthwhile to compare the different mutant phenotypes. For example, signaling by platelet-derived growth factor (PDGF) B and its receptor PDGFRβ is essential for the proliferation, chemotactic guidance, and association of PCs/VSMCs. Studies in cultured cells have established that this pathway synergistically cooperates with integrins.^26,27 However, this crosstalk involves integrin αvβ3 rather than complexes with β1, and we found that Itgb1^Pdgfrb-Cre mutants do not recapitulate the dramatic reduction of PCs/VSMCs caused by the inactivation of the Pdgfb or Pdgfrb genes.³

We have shown previously that that mural cells require ephrin-B2, a small transmembrane protein and ligand for Eph family receptor tyrosine kinases, for their correct association with capillaries and small caliber arteries and veins.¹⁷ Even though cultured ephrin-B2–deficient aortic SMCs display prominent focal adhesion defects, the accelerated but random motility of these cells appears very distinct from the comparably static Itgb1 KO cells. Thus, mural cell-specific integrin β1 mutants fall into a separate phenotypic category characterized by compromised PC and VSMCs spreading, migration, and differentiation.

Redundancy Versus Functional Specificity of Integrin Receptors

Several mutant mice lacking individual integrin α subunits develop vascular defects, suggesting that these molecules may partner with β1 in PCs and VSMCs. Integrin α5β1 is strongly expressed in SMCs, and the knockout of the Itga5 gene encoding the α5 subunit reproduces the very severe vascular defects seen in embryos lacking fibronectin, the major ligand of α5β1.^11,28 The midgestation lethality of Itga5 embryos is likely to reflect a combination of problems affecting the heart, the endothelium, and, possibly, mural cells. Tissue-specific loss-of-function studies will be required to identify the roles of integrin α5 in individual cell types. Similar to α5, the majority of Itga4 (integrin α4) mutants die at midgestation, which has been attributed to failed chorioallantoic fusion.²⁹ A fraction of mutants surviving up to E14.5 displays defective distribution of PCs/VSMCs in the cranial vasculature.³⁰ Adhesion and motility of cultured Itga4 cells was also reduced, similar to Itgb1^KO SMCs.^30,31 It also has been shown that the laminin receptor integrin α7β1 is expressed in SMCs, and cerebrovascular hemorrhaging in the knockout mice has been attributed to VSMC defects.^32,33

Given that integrin β1 forms functional complexes with all the subunits mentioned above, mediates adhesion to fibronectin, laminin, and collagen substrates, and also controls fibronectin fibrillogenesis in the vessel wall, it is surprising that VSMC survival or proliferation are not disrupted in Itgb1^Pdgfrb-Cre mutants. Similarly, mutant PCs are compromised in their ability to spread and support ECs but are actually present in increased numbers. These data suggest that other integrins, such as αvβ3 or αvβ5, provide sufficient adhesion for necessary promitotic and antiapoptotic signals. Indeed, the integrins αvβ3 or αvβ5 are expressed by SMCs and can bind a set of ECM molecules that partially overlaps with β1-containing receptor complexes.³⁴

Integrin β1 and the Synthetic VSMC Phenotype

Changes in the matrix environment or the expression, cell surface presentation, binding, or signaling properties of integrins may cause or contribute to pathological changes in the vasculature. For example, it has been shown that laminin and type I or type IV collagens help to maintain a differentiated, contractile phenotype of cultured arterial SMCs, whereas the cell attachment (RGD) motif of fibronectin has the opposite effect.^35–38 Experimentally induced vascular injury reduces the local expression and activity of β1 integrins in vascular cells, whereas αvβ3 or αvβ5 and the corresponding matrix substrates are upregulated.^5,39–41 Moreover, β3 integrins favor a poorly differentiated and highly motile SMC phenotype, and knockout mice are protected against pathological VSMC migration and neointima formation.^5,40–44

The sum of these findings suggests that different integrin complexes may have opposite roles in the regulation of the SMC phenotype. The defects in Itgb1^Pdgfrb-Cre mutant mice directly confirms that integrin β1 is essential for VSMC morphology, differentiation, and function and may provide useful leads for future research investigating tissue repair and vascular regeneration processes.