Objective Prenatal deletion of the type II TGF-β receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-β signaling and blockade of TGF-β signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy. Approach and Results We generated mice with either of two tamoxifen-inducible SMC-specific Cre (SMC-CreERT2) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreERT2 alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-β signaling and reproduced some of the gene expression abnormalities detected in vivo. Conclusions SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-β signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacologic blockade of TGF-β signaling in humans could cause aortic disease rather than prevent it.

BackgroundMarfan syndrome (MFS) is caused by mutations in the gene encoding fibrillin‐1 (FBN1); however, the mechanisms through which fibrillin‐1 deficiency causes MFS‐associated aortopathy are uncertain. Recently, attention was focused on the hypothesis that MFS‐associated aortopathy is caused by increased transforming growth factor‐β (TGF‐β) signaling in aortic medial smooth muscle cells (SMC). However, there are many reasons to doubt that TGF‐β signaling drives MFS‐associated aortopathy. We used a mouse model to test whether SMC TGF‐β signaling is perturbed by a fibrillin‐1 variant that causes MFS and whether blockade of SMC TGF‐β signaling prevents MFS‐associated aortopathy.Methods and Results MFS mice (Fbn1 C1039G/+ genotype) were genetically modified to allow postnatal SMC‐specific deletion of the type II TGF‐β receptor (TBRII; essential for physiologic TGF‐β signaling). In young MFS mice with and without superimposed deletion of SMC‐TBRII, we measured aortic dimensions, histopathology, activation of aortic SMC TGF‐β signaling pathways, and changes in aortic SMC gene expression. Young Fbn1 C1039G/+ mice had ascending aortic dilation and significant disruption of aortic medial architecture. Both aortic dilation and disrupted medial architecture were exacerbated by superimposed deletion of TBRII. TGF‐β signaling was unaltered in aortic SMC of young MFS mice; however, SMC‐specific deletion of TBRII in Fbn1 C1039G/+ mice significantly decreased activation of SMC TGF‐β signaling pathways.ConclusionsIn young Fbn1 C1039G/+ mice, aortopathy develops in the absence of detectable alterations in SMC TGF‐β signaling. Loss of physiologic SMC TGF‐β signaling exacerbates MFS‐associated aortopathy. Our data support a protective role for SMC TGF‐β signaling during early development of MFS‐associated aortopathy.

Objective The role of TGF-β signaling in abdominal aortic aneurysm (AAA) formation is controversial. Others reported that systemic blockade of TGF-β by neutralizing antibodies accelerated AAA development in angiotensin II-infused mice. This result is consistent with other studies suggesting that TGF-β signaling prevents AAA. Development of a therapy for AAA that exploits the protective actions of TGF-β would be facilitated by identification of the mechanisms through which TGF-β prevents AAA. We hypothesized that TGF-β signaling prevents AAA by its actions on aortic medial smooth muscle cells. Approach and Results We compared the prevalence, severity, and histopathology of angiotensin II-induced AAA among control mice (no TGF-β blockade), mice with antibody-mediated systemic neutralization of TGF-β, and mice with genetically based smooth muscle-specific loss of TGF-β signaling. Surprisingly, we found that systemic—but not smooth muscle-specific—TGF-β blockade significantly increased the prevalence of AAA, and tended to increase AAA severity, adventitial thickening and aortic wall macrophage accumulation. In contrast, abdominal aortas of mice with smooth muscle-specific loss of TGF-β signaling differed from controls only in having a thinner media. We examined thoracic aortas of the same mice. Here we found that smooth muscle-specific loss of Tgfbr2—but not systemic TGF-β neutralization—significantly accelerated development of aortic pathology, including increased prevalence of intramural hematomas, medial thinning, and adventitial thickening. Conclusion Our results suggest that TGF-β signaling prevents both abdominal and thoracic aneurysmal disease but does so by distinct mechanisms. Smooth muscle-extrinsic signaling protects the abdominal aorta and smooth muscle-intrinsic signaling protects the thoracic aorta.

The unique contributions of connexin (Cx)37 and Cx40, gap junction-forming proteins that are coexpressed in vascular endothelium, to the recovery of tissues from ischemic injury are unknown. We recently reported that Cx37-deficient (Cx37(-/-)) animals recovered ischemic hindlimb function more quickly and to a greater extent than wild-type (WT) or Cx40(-/-) animals, suggesting that Cx37 limits recovery in the WT animal. Here, we tested the hypothesis that enhanced angiogenesis, arteriogenesis, and vasculogenesis contribute to improved postischemic hindlimb recovery in Cx37(-/-) animals. Ischemia was induced unilaterally in the hindlimbs of WT or Cx37(-/-) mice (isoflurane anesthesia). Postsurgical limb appearance, use, and perfusion were documented during recovery, and the number (and size) of large and small vessels was determined. Native collateral number, predominantly established during embryonic development (vasculogenesis), was also determined in the pial circulation. Both microvascular density in the gastrocnemius of the ischemic limb (an angiogenic field) and the number and tortuosity of larger vessels in the gracilis vasculature (an arteriogenic field) were increased in Cx37(-/-) animals compared with WT animals. Cx37(-/-) mice also had an increased (vs. WT) number of collateral vessels in the pial circulation. These findings suggest that in Cx37(-/-) animals, improved recovery of the ischemic hindlimb involves enhanced vasculogenesis, resulting in increased numbers of collaterals in the hindlimb (and pial circulations) and more extensive collateral remodeling and angiogenesis. These results are consistent with Cx37 exerting a growth-suppressive effect in the vasculature that limits embryonic vasculogenesis as well as arteriogenic and angiogenic responses to ischemic injury in the adult animal.

Background/Aims: Ischemia induced by large-vessel obstruction or vascular injury induces a complex cascade of vasodilatory, remodeling and inflammatory pathways; coordination of these processes by vascular endothelium is likely to involve endothelial gap junctions. Vascular endothelium predominantly expresses two connexin (Cx) isoforms: Cx37 and Cx40. The relevance of these Cxs to postischemic limb recovery remains unclear. Methods: In this study, we use a well-established, severe femoral-saphenous artery-vein pair resection model of unilateral hindlimb ischemia to test the relevance of Cx37 and Cx40 to postischemic tissue survival and recovery of limb perfusion. Results: Cx40-deficient animals (Cx40–/–) experienced a severe reduction in limb perfusion relative to wild-type (WT) animals and exhibited profound and rapid failure of ischemic limb survival. By contrast, the deficit in limb perfusion was less severe in Cx37-ablated (Cx37–/–) animals compared to WT, corresponding with more rapid recovery of limb appearance and use. These results demonstrate that Cx40 is necessary for postischemic limb survival and reperfusion, whereas Cx37 deletion reduces the extent of ischemia in the same model. Conclusion: In summary, we present evidence demonstrating that Cx37 and Cx40 uniquely regulate postischemic limb perfusion, altering the severity of ischemic insult and consequent postischemic survival.