Amy Mohan

Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II–induced aortic aneurysms

Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe−/− mice, we show that Apoe−/−Ppia−/− mice are completely protected from AngII–induced AAA formation, in contrast to Apoe−/−Ppia+/+ mice. Apoe−/−Ppia−/− mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.

Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation

Background— Oxidative stress, generated by excessive reactive oxygen species, promotes cardiovascular disease. Cyclophilin A (CyPA) is a 20-kDa chaperone protein secreted from vascular smooth muscle cells (VSMCs) in response to reactive oxygen species that stimulates VSMC proliferation and inflammatory cell migration in vitro; however, the role CyPA plays in vascular function in vivo remains unknown. Methods and Results— We tested the hypothesis that CyPA contributes to vascular remodeling by analyzing the response to complete carotid ligation in CyPA knockout mice, wild-type mice, and mice that overexpress CyPA in VSMC (VSMC-Tg). After carotid ligation, CyPA expression in vessels of wild-type mice increased dramatically and was significantly greater in VSMC-Tg mice. Reactive oxygen species–induced secretion of CyPA from mouse VSMCs correlated significantly with intracellular CyPA expression. Intimal and medial hyperplasia correlated significantly with CyPA expression after 2 weeks of carotid ligation, with marked decreases in CyPA knockout mice and increases in VSMC-Tg mice. Inflammatory cell migration into the intima was significantly reduced in CyPA knockout mice and increased in VSMC-Tg mice. Additionally, VSMC proliferation assessed by Ki67+ cells was significantly less in CyPA knockout mice and was increased in VSMC-Tg mice. The importance of CyPA for intimal and medial thickening was shown by strong correlations between CyPA expression and the number of both inflammatory cells and proliferating VSMCs in vivo and in vitro. Conclusions— In response to low flow, CyPA plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating flow-mediated vascular remodeling and intima formation.

Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E–deficient mice

Cyclophilin A promotes atherosclerosis in part by inducing reactive oxygen species and promoting endothelial cell apoptosis and macrophage recruitment into lesions.

Axl, A Receptor Tyrosine Kinase, Mediates Flow-Induced Vascular Remodeling

Intima-media thickening (IMT) in response to hemodynamic stress is a physiological process that requires coordinated signaling among endothelial, inflammatory, and vascular smooth muscle cells (VSMC). Axl, a receptor tyrosine kinase, whose ligand is Gas6, is highly induced in VSMC after carotid injury. Because Axl regulates cell migration, phagocytosis and apoptosis, we hypothesized that Axl would play a role in IMT. Vascular remodeling in mice deficient in Axl (Axl−/−) and wild-type littermates (Axl+/+) was induced by ligation of the left carotid artery (LCA) branches maintaining flow via the left occipital artery. Both genotypes had similar baseline hemodynamic parameters and carotid artery structure. Partial ligation altered blood flow equally in both genotypes: increased by 60% in the right carotid artery (RCA) and decreased by 80% in the LCA. There were no significant differences in RCA remodeling between genotypes. However, in the LCA Axl−/− developed significantly smaller intima+media compared with Axl+/+ (31±4 versus 42±6×10−6 &mgr;m3, respectively). Quantitative immunohistochemistry of Axl−/− LCA showed increased apoptosis compared with Axl+/+ (5-fold). As expected, p-Akt was decreased in Axl−/−, whereas there was no difference in Gas6 expression. Cell composition also changed significantly, with increases in CD45+ cells and decreases in VSMC, macrophages, and neutrophils in Axl−/− compared with Axl+/+. These data demonstrate an important role for Axl in flow-dependent remodeling by regulating vascular apoptosis and vascular inflammation.

Role of Nuclear Ca2+/Calmodulin-Stimulated Phosphodiesterase 1A in Vascular Smooth Muscle Cell Growth and Survival

In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated “contractile” phenotype to a dedifferentiated “synthetic” one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca2+-calmodulin–stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial “contractile” VSMCs but is nuclear in neointimal “synthetic” VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the “contractile” phenotype, whereas nuclear PDE1A is with the “synthetic” phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis.

Hydrogen Peroxide Activates the Gas6-Axl Pathway in Vascular Smooth Muscle Cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H2O2 might activate Axl by promoting autophosphorylation. H2O2 rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H2O2 was concentration-dependent with EC50 of ∼500 μm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H2O2-induced Axl phosphorylation by 50%. Axl phosphorylation by H2O2 was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H2O2, and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H2O2in Axl+/+ mouse aorta but significantly inhibited in Axl-/- aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl-/- mice compared with Axl+/+ mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.

Flow Antagonizes TNF-α Signaling in Endothelial Cells by Inhibiting Caspase-Dependent PKCζ Processing

Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-&agr; signaling by preventing activation of JNK. Recently, PKC&zgr; was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKC&zgr; activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKC&zgr; is cleaved by caspases after TNF-&agr; stimulation to generate a 50-kDa truncated form (CAT&zgr;, catalytic domain of PKC&zgr;) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-&agr;–mediated PKC&zgr; cleavage and thereby CAT&zgr; formation. We found that PKC&zgr; activity was required for TNF-&agr;–mediated JNK and caspase-3 activation in ECs. PKC&zgr; was rapidly cleaved to generate CAT&zgr; in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-&agr;. This truncated form of PKC&zgr; enhanced JNK and caspase-3 activation. Interestingly, PKC&zgr; cleavage was prevented by inhibitors of PKC&zgr;, JNK, and caspase activities, suggesting that these enzymes, via regulating CAT&zgr; formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKC&zgr; and caspase-3 activation. These results define a novel role for PKC&zgr; as a shared signaling mediator for flow and TNF-&agr;, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.

Cyclophilin A Promotes Cardiac Hypertrophy in Apolipoprotein E–Deficient Mice

Objective—Cyclophilin A (CyPA, encoded by Ppia) is a proinflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (Ang II)–induced reactive oxygen species (ROS) production in the aortas of apolipoprotein E (Apoe)−/− mice. In this study, we sought to evaluate the role of CyPA in Ang II–induced cardiac hypertrophy. Methods and Results—Cardiac hypertrophy was not significantly different between Ppia+/+ and Ppia−/− mice infused with Ang II (1000 ng/min per kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe−/− mice. In contrast to Apoe−/− mice, Apoe−/−Ppia−/− mice exhibited significantly less Ang II–induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. Ang II–induced ROS production, cardiac fibroblast proliferation, and cardiac fibroblast migration were markedly decreased in Apoe−/−Ppia−/− cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. Conclusion—CyPA is required for Ang II–mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.

Angiotensin II Type 2 Receptor Expression After Vascular Injury: Differing Effects of Angiotensin-Converting Enzyme Inhibition and Angiotensin Receptor Blockade

It has been suggested that the effects of angiotensin II type 1 receptor (AT1R) blockers are in part because of angiotensin II type 2 receptor (AT2R) signaling. Interactions between the AT2R and kinins modulate cardiovascular function. Because AT2R expression increases after vascular injury, we hypothesized that the effects on vascular remodeling of the AT1R blocker valsartan and the ACE inhibitor benazepril require AT2R signaling through the bradykinin 1 and 2 receptors (B1R and B2R). To test this hypothesis, Brown Norway rats were assigned to 8 treatments (n=16): valsartan, valsartan+PD123319 (AT2R inhibitor), valsartan+des-arg9-[Leu8]-bradykinin (B1R inhibitor), valsartan+HOE140 (B2R inhibitor), benazepril, benazepril+HOE140, amlodipine, and vehicle. After 1 week of treatment, carotid balloon injury was performed. Two weeks later, carotids were harvested for morphometry and analysis of receptor expression by immunohistochemistry and Western blotting. Valsartan and benazepril significantly reduced the intima:media ratio compared with vehicle. Blockade of AT2R, B1R, or B2R in the presence of valsartan prevented the reduction seen with valsartan alone. B2R blockade inhibited the effect of benazepril. Injury increased AT1R, AT2R, B1R, and B2R expression. Treatment with valsartan but not benazepril significantly increased intima AT2R expression 2-fold compared with vehicle, which was not reversed by inhibition of AT2R, B1R, and B2R. Functionally, valsartan increased intimal cGMP levels compared with vehicle, and this increase was inhibited by blocking the AT2R, B1R, and B2R. Results suggest that AT2R expression and increased cGMP represent a molecular mechanism that differentiates AT1R blockers, such as valsartan, from angiotensin-converting enzyme inhibitors like benazepril.

G-Protein–Coupled Receptor Kinase Interacting Protein-1 Is Required for Pulmonary Vascular Development

Background— The G-protein–coupled receptor kinase interacting protein-1 (GIT1) is a multidomain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both G-protein–coupled receptors and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date. Methods and Results— To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mouse. GIT1 knockout mice exhibited ≈60% perinatal mortality. Pathological examination showed that the major abnormality in GIT1 knockout mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Given that vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells. Because activation of phospholipase-Cγ (PLCγ) and extracellular signal-regulated kinases 1/2 (ERK1/2) by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCγ and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCγ and ERK1/2. PLCγ and ERK1/2 activity in lungs from GIT1 knockout mice was reduced postnatally. Conclusions— Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCγ and ERK1/2 activation.