UCLA

Extracellular matrix (ECM) organization is critical for the maintenance of vascular integrity and for regulating biochemical signals that are instructive during the development and maturation of vascular networks. Cell-ECM interactions are mediated by a class of transmembrane glycoprotein known as integrins, which form heterodimeric alpha/beta; receptors for the ECM. Beta1 integrin (Beta1) is ubiquitously and constitutively expressed in the vasculature where it binds to numerous proteins in ECM such as fibronectin (FN), collagen and fibrillin (FBLN). To evaluate its specific biological function during vascular development, we used the CRE-lox system to delete beta1 in vascular smooth muscle cells (vSMCs). Homozygous deletion was embryonic lethal by E18.5 and resulted in severe defects in the cardiovascular system. Early expression of CRE recombinase also resulted in beta1 deletion in cardiomyocytes. As a result, smooth muscle beta1 knockout (SMB1KO) embryos demonstrated delayed maturation of the myocardium, and loose compaction of the cells in the trabeculum. However, the predominating defect and the focus of a majority of this work was related to site-specific aneurysm formation at the bifurcations of the aortic arch derivatives.

Assessment of the ECM of the vasculature by immunofluorescence showed lack of lamellar organization that was particularly more pronounced at sites of aneurysms. Evaluation of the 3D architecture by electron microscopy revealed dysmorphic vSMCs with aberrant cell-matrix connections, and elastic fiber fragmentation. Upon further investigation, we found a selective decrease in the mRNA levels of ECM proteins specific to the elastin/microfibril system (i.e. FBLN-1, lysyl oxidase and fibulin-5). Consistent with this, protein levels of elastin (ELN) and FBLN-1 were decreased in the aortic arches of SMB1KO embryos, indicating a profound disruption of elastic lamina formation at the vascular wall in the absence of beta1.

To further understand the mechanistic contribution of beta1 in complex ECM assembly, we adopted an in vitro model using immortalized smooth muscle cells (IMSMCs). Analysis of ECM assembly showed diminished fibril formation of FN, FBLN1 and ELN in the absence of beta1, despite no differences in the levels of secreted FN and FBLN1 protein. However, when cultured in an assembled fibrin network, B1KO IMSMCs were able to partially rescue this defect, forming large-bundled fibers with non-uniform dimensions. Control IMSMCs, on the other hand, formed a regular pattern of ECM fibrils.

With respect to external stimulation, unlike control IMSMCs, B1KO cells were unresponsive to the addition of tissue transglutaminase (TG2) which enzymatically crosslinks FN fibrils. Additionally, loss of beta1 rendered IMSMCs unresponsive to growth stimulation by TGF-beta1, FGF-2 and PDGF-beta;. Both in vivo and in vitro results are suggestive of a key regulatory role for beta1 in elastic fiber homeostasis, and growth factor stimulation, demonstrating that beta1 is not only critical for maintaining vasculature integrity, but its connection to the ECM is a necessary mediator of matrix expression, assembly and remodeling.