Arterial stiffness and cardiac dysfunction in Hutchinson-Gilford Progeria Syndrome corrected by inhibition of lysyl oxidase.

Arterial stiffening and cardiac dysfunction are hallmarks of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), but the molecular regulators remain unknown. Here, we show that the LaminAG609G mouse model of HGPS recapitulates the premature arterial stiffening and early diastolic dysfunction seen in human HGPS. Lysyl oxidase (LOX) is up-regulated in the arteries of these mice, and treatment with the LOX inhibitor, ß-aminopropionitrile, improves arterial mechanics and cardiac function. Genome-wide and mechanistic analysis revealed reduced expression of the LOX-regulator, miR-145, in HGPS arteries, and forced expression of miR-145 restores normal LOX gene expression in HGPS smooth muscle cells. LOX abundance is also increased in the carotid arteries of aged wild-type mice, but its spatial expression differs from HGPS and its up-regulation is independent of changes in miR-145 abundance. Our results show that miR-145 is selectively misregulated in HGPS and that the consequent up-regulation of LOX is causal for premature arterial stiffening and cardiac dysfunction.

MMP12 Deletion Preferentially Attenuates Axial Stiffening of Aging Arteries.

Arterial stiffening is a hallmark of aging, but how aging affects the arterial response to pressure is still not completely understood, especially with regard to specific matrix metalloproteinases (MMPs). Here, we performed biaxial inflation-extension tests on C57BL/6 mice to study the effects of age and MMP12, a major arterial elastase, on arterial biomechanics. Aging from 2 to 24 months leads to both circumferential and axial stiffening with stretch, and these changes are associated with an increased wall thickness, a decreased inner radius-wall thickness ratio, and a decreased in vivo axial stretch. Analysis of in vivo stretch and stress-stretch curves with arteries from age- and sex-matched wild-type (WT) and MMP12-null arteries demonstrates that MMP12 deletion attenuates age-dependent arterial stiffening, mostly in the axial direction. MMP12 deletion also prevents the aging-associated decrease in the in vivo stretch and, in general, leads to an axial mechanics phenotype characteristic of much younger mice. Circumferential arterial mechanics were much less affected by deletion of MMP12. We conclude that the induction of MMP12 during aging preferentially promotes axial arterial stiffening.

Cardiovascular protection in females linked to estrogen-dependent inhibition of arterial stiffening and macrophage MMP12.

Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL-induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.

Matrix metalloproteinase-12 is an essential mediator of acute and chronic arterial stiffening.

Arterial stiffening is a hallmark of aging and risk factor for cardiovascular disease, yet its regulation is poorly understood. Here we use mouse modeling to show that matrix metalloproteinase-12 (MMP12), a potent elastase, is essential for acute and chronic arterial stiffening. MMP12 was induced in arterial smooth muscle cells (SMCs) after acute vascular injury. As determined by genome-wide analysis, the magnitude of its gene induction exceeded that of all other MMPs as well as those of the fibrillar collagens and lysyl oxidases, other common regulators of tissue stiffness. A preferential induction of SMC MMP12, without comparable effect on collagen abundance or structure, was also seen during chronic arterial stiffening with age. In both settings, deletion of MMP12 reduced elastin degradation and blocked arterial stiffening as assessed by atomic force microscopy and immunostaining for stiffness-regulated molecular markers. Isolated MMP12-null SMCs sense extracellular stiffness normally, indicating that MMP12 causes arterial stiffening by remodeling the SMC microenvironment rather than affecting the mechanoresponsiveness of the cells themselves. In human aortic samples, MMP12 levels strongly correlate with markers of SMC stiffness. We conclude that MMP12 causes arterial stiffening in mice and suggest that it functions similarly in humans.

miR-221/222 compensates for Skp2-mediated p27 degradation and is a primary target of cell cycle regulation by prostacyclin and cAMP.

p27(kip1) (p27) is a cdk-inhibitory protein with an important role in the proliferation of many cell types. SCF(Skp2) is the best studied regulator of p27 levels, but Skp2-mediated p27 degradation is not essential in vivo or in vitro. The molecular pathway that compensates for loss of Skp2-mediated p27 degradation has remained elusive. Here, we combine vascular injury in the mouse with genome-wide profiling to search for regulators of p27 during cell cycling in vivo. This approach, confirmed by RT-qPCR and mechanistic analysis in primary cells, identified miR-221/222 as a compensatory regulator of p27. The expression of miR221/222 is sensitive to proteasome inhibition with MG132 suggesting a link between p27 regulation by miRs and the proteasome. We then examined the roles of miR-221/222 and Skp2 in cell cycle inhibition by prostacyclin (PGI(2)), a potent cell cycle inhibitor acting through p27. PGI(2) inhibited both Skp2 and miR221/222 expression, but epistasis, ectopic expression, and time course experiments showed that miR-221/222, rather than Skp2, was the primary target of PGI(2). PGI(2) activates Gs to increase cAMP, and increasing intracellular cAMP phenocopies the effect of PGI(2) on p27, miR-221/222, and mitogenesis. We conclude that miR-221/222 compensates for loss of Skp2-mediated p27 degradation during cell cycling, contributes to proteasome-dependent G1 phase regulation of p27, and accounts for the anti-mitogenic effect of cAMP during growth inhibition.

Cardiovascular protection by ApoE and ApoE-HDL linked to suppression of ECM gene expression and arterial stiffening.

Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein (apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.

Cell-type- and cell-cycle-specific anti-mitogenesis by cicaprost.

Stents eluting anti-proliferative drugs limit restenosis, but drugs commonly used to date are relatively non-specific cytostatic agents which inhibit proliferation of intimal endothelial cells as well as medial smooth muscle cells and may thereby contribute to the clinical complications associated with angioplasty. In an effort to identify a more specific anti-proliferative agent, we compared the effects of rapamycin to those of cicaprost, a mimetic of the naturally occurring anti-mitogen, PGI(2). Rapamycin and cicaprost were both strongly anti-mitogenic in vascular smooth muscle cells (VSMCs). But unlike rapamycin, cicaprost did not inhibit mitogenesis in aortic endothelial cells even when used at concentrations >10-fold higher than its ED(50) for VSMCs. Similarly, both rapamycin and cicaprost have been reported to regulate levels of the cdk inhibitor, p27(kip1). But rapamycin remained anti-mitogenic in p27(kip1)-null VSMCs whereas the anti-mitogenic effect of cicaprost was completely dependent on p27(kip1). We conclude that stable PGI(2) mimetics may be highly specific inhibitors of p27(kip1)-dependent VSMC proliferation after vascular injury.

Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.

BACKGROUND: A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle. RESULTS: We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo. CONCLUSIONS: Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.

Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells.

High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031-1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27(kip1) in vascular SMCs. p27(kip1) messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27(kip1), was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury.

Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation.

HDL and its associated apo, APOE, inhibit S-phase entry of murine aortic smooth muscle cells. We report here that the antimitogenic effect of APOE maps to the N-terminal receptor-binding domain, that APOE and its N-terminal domain inhibit activation of the cyclin A promoter, and that these effects involve both pocket protein-dependent and independent pathways. These antimitogenic effects closely resemble those seen in response to activation of the prostacyclin receptor IP. Indeed, we found that HDL and APOE suppress aortic smooth muscle cell cycle progression by stimulating Cox-2 expression, leading to prostacyclin synthesis and an IP-dependent inhibition of the cyclin A gene. Similar results were detected in human aortic smooth muscle cells and in vivo using mice overexpressing APOE. Our results identify the Cox-2 gene as a target of APOE signaling, link HDL and APOE to IP action, and describe a potential new basis for the cardioprotective effect of HDL and APOE.