ß1-integrin is essential for vasoregulation and smooth muscle survival in vivo.

OBJECTIVE: Integrins contribute to vascular morphogenesis through regulation of adhesion and assembly of the extracellular matrix. However, the role of ß1-integrin in the mature vascular wall is less clear. APPROACH AND RESULTS: We sought to determine the function of ß1-integrin in mature smooth muscle cells in vivo using a loss of function approach by crossing a tamoxifen-inducible sm22αCre line to a floxed ß1-integrin transgenic line. Adult mice lacking smooth muscle ß1-integrin survived only 10 weeks post induction. The deletion of ß1-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 ß1-integrin. CONCLUSIONS: This study reveals an essential function of ß1-integrin in the maintenance of vasomotor control and highlights a critical role for ß1-integrin in vascular, but not visceral, smooth muscle survival.

An essential requirement for ß1 integrin in the assembly of extracellular matrix proteins within the vascular wall.

ß1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of ß1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that ß1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of ß1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for ß1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that ß1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of ß1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.

Assessing mechanisms of GPIHBP1 and lipoprotein lipase movement across endothelial cells.

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.

Vascular remodeling of the vitelline artery initiates extravascular emergence of hematopoietic clusters.

The vitelline artery is a temporary structure that undergoes extensive remodeling during midgestation to eventually become the superior mesenteric artery (also called the cranial mesenteric artery, in the mouse). Here we show that, during this remodeling process, large clusters of hematopoietic progenitors emerge via extravascular budding and form structures that resemble previously described mesenteric blood islands. We demonstrate through fate mapping of vascular endothelium that these mesenteric blood islands are derived from the endothelium of the vitelline artery. We further show that the vitelline arterial endothelium and subsequent blood island structures originate from a lateral plate mesodermal population. Lineage tracing of the lateral plate mesoderm demonstrates contribution to all hemogenic vascular beds in the embryo, and eventually, all hematopoietic cells in the adult. The intraembryonic hematopoietic cell clusters contain viable, proliferative cells that exhibit hematopoietic stem cell markers and are able to further differentiate into myeloid and erythroid lineages. Vitelline artery-derived hematopoietic progenitor clusters appear between embryonic day 10 and embryonic day 10.75 in the caudal half of the midgut mesentery, but by embryonic day 11.0 are sporadically found on the cranial side of the midgut, thus suggesting possible extravascular migration aided by midgut rotation.

When Cre-mediated recombination in mice does not result in protein loss.

Cre/loxP recombination enables cellular specificity and, in the case of inducible systems, temporal control of genomic deletions. Here we used a SM22α tamoxifen-inducible Cre line to inactivate ß1 integrin in adult smooth muscle. Interestingly, analysis of two distinct ß1 loxP transgenic mice revealed vastly different outcomes after ß1 integrin deletion. Lethality occurred 4 weeks postinduction in one Cre/loxP line, while no apparent phenotype was seen in the other line. Genetic analysis revealed appropriate DNA excision in both cases; however, differences were found in the degree of protein loss with absolutely no change in protein levels in the model that lacked a phenotype. Seeking to understand protein persistence despite appropriate recombination, we first validated the flox allele using a constitutive Cre line and demonstrated its ability to mediate effective protein inactivation. We then examined the possibility of heterozygous cell selection, protein turnover, and deletion efficiency with no success for explaining the phenotype. Finally, we documented the presence of the Cre-recombination episomal product, which persisted in tissue samples with no protein loss. The product was only noted in cells with low proliferative capacity. These findings highlight the potential for protein expression from the products of Cre-recombinase excised genes, particularly when deletion occurs in low turnover populations.

Beta1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism.

Maintenance of single-layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of beta1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal-shaped endothelial cells with dysregulated levels and mislocalization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). beta1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in beta1-deficient vessels. Combined, our findings demonstrate that beta1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation.

Fate tracing reveals the endothelial origin of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) originate within the aortic-gonado-mesonephros (AGM) region of the midgestation embryo, but the cell type responsible for their emergence is unknown since critical hematopoietic factors are expressed in both the AGM endothelium and its underlying mesenchyme. Here we employ a temporally restricted genetic tracing strategy to selectively label the endothelium, and separately its underlying mesenchyme, during AGM development. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the endothelium is capable of HSC emergence. The endothelial progeny migrate to the fetal liver, and later to the bone marrow, and are capable of expansion, self-renewal, and multilineage hematopoietic differentiation. HSC capacity is exclusively endothelial, as ex vivo analyses demonstrate lack of VE-cadherin Cre induction in circulating and fetal liver hematopoietic populations. Moreover, AGM mesenchyme, as selectively traced via a myocardin Cre line, is incapable of hematopoiesis. Our genetic tracing strategy therefore reveals an endothelial origin of HSCs.