The effect of four loading intravitreal aflibercept injections on macular fluid in treatment-naïve neovascular age-related macular degeneration.

PURPOSE: To evaluate the effect of four versus three loading aflibercept injections on macular fluid resolution and visual acuity (VA) in exudative neovascular AMD (nAMD). METHODS: Multicentre, retrospective cohort study of treatment naïve nAMD eyes undergoing 3 versus 4 loading doses of aflibercept. Change in VA and fluid resolution on optical coherence tomography (OCT), were evaluated at 8 weeks post loading. The primary outcome was proportion of patients with no intraretinal (IRF) and/or subretinal (SRF) fluid at central 1 mm and whole macula at 8 weeks after loading. Data were summarised with mean ± SD for continuous variables, and n (%) for categorical variables. RESULTS: Data from 995 patients was analysed (355 patients - 4 loading doses and 640-3 loading doses). At 8 weeks post 4 loading doses proportion of eyes with neither IRF nor SRF, no IRF and no SRF were 62.8%, 88.7% and 79.2% at fovea versus 56.1%, 87.9% and 69.9% in the whole macula, respectively. Fluid resolution at both fovea and macula were significantly higher in eyes with 4 loading injections versus 3 (p = 0.0001). The mean VA change was +4.0 (±11.3) and +5.4(±13.3) letters for 3 and 4 loading doses groups (p = 0.09). CONCLUSION: Four loading dose injections of aflibercept results in higher proportion of eyes with total fluid resolution in the central subfield and total macular scan when compared to those receiving 3 loading dose injections at 8 weeks post loading phase. However, the better drying effect of 4th loading dose does not translate into better short-term VA outcomes.

Platelet olfactory receptor activation limits platelet reactivity and growth of aortic aneurysms.

As blood transitions from steady laminar flow (S-flow) in healthy arteries to disturbed flow (D-flow) in aneurysmal arteries, platelets are subjected to external forces. Biomechanical platelet activation is incompletely understood and is a potential mechanism behind antiplatelet medication resistance. Although it has been demonstrated that antiplatelet drugs suppress the growth of abdominal aortic aneurysms (AAA) in patients, we found that a certain degree of platelet reactivity persisted in spite of aspirin therapy, urging us to consider additional antiplatelet therapeutic targets. Transcriptomic profiling of platelets from patients with AAA revealed upregulation of a signal transduction pathway common to olfactory receptors, and this was explored as a mediator of AAA progression. Healthy platelets subjected to D-flow ex vivo, platelets from patients with AAA, and platelets in murine models of AAA demonstrated increased membrane olfactory receptor 2L13 (OR2L13) expression. A drug screen identified a molecule activating platelet OR2L13, which limited both biochemical and biomechanical platelet activation as well as AAA growth. This observation was further supported by selective deletion of the OR2L13 ortholog in a murine model of AAA that accelerated aortic aneurysm growth and rupture. These studies revealed that olfactory receptors regulate platelet activation in AAA and aneurysmal progression through platelet-derived mediators of aortic remodeling.

Sildenafil (Viagra) Aggravates the Development of Experimental Abdominal Aortic Aneurysm.

Background cGMP-hydrolyzing phosphodiesterase type 5 (PDE5) regulates vascular smooth muscle cell (SMC) contraction by antagonizing cGMP-dependent protein kinase I (PKGI)-dependent SMC relaxation. SMC contractile dysfunction is implicated in the pathogenesis of aortic aneurysm. PDE5 inhibitors have been used for treating erectile dysfunction, such as drug Viagra (sildenafil). However, a few clinical cases have reported the association of Viagra usage with aortic dissection, and reduced PDE5A expression was found in human aortic aneurysm tissues. Therefore, we aimed to investigate the effect of sildenafil on experimental abdominal aortic aneurysm (AAA), the most common form of aortic aneurysm in elderly men. Methods and Results AAA was induced in C57BL/6J male mice by periaortic elastase in combination with blocking elastin/collagen formation via 3-aminopropionitrile fumarate salt for 35 days. PDE5A protein levels detected by immunostaining were significantly reduced in mouse AAA. Sildenafil application in drinking water significantly aggravated aortic wall dilation and elastin degradation with pre-existing moderate AAA. The phosphorylation level of myosin light chain 2 at Ser19, a biochemical marker of SMC contraction, was significantly reduced by sildenafil in AAA. Proximity ligation assay further revealed that the interaction between cGMP and PKGI was significantly increased by sildenafil in AAA, suggesting an elevation of PKGI activation in AAA. Conclusions Sildenafil treatment aggravated the degradation of elastin fibers and progression of experimental AAA by dysregulating cGMP and contractile signaling in SMCs. Our findings may raise the caution of clinical usage of Viagra in aneurysmal patients.

Reduced Notch1 Cleavage Promotes the Development of Pulmonary Hypertension.

Clinical trials of Dll4 (Delta-like 4) neutralizing antibodies (Dll4nAbs) in cancer patients are ongoing. Surprisingly, pulmonary hypertension (PH) occurs in 14% to 18% of patients treated with Dll4nAbs, but the mechanisms have not been studied. Here, PH progression was measured in mice treated with Dll4nAbs. We detected Notch signaling in lung tissues and analyzed pulmonary vascular permeability and inflammation. Notch target gene array was performed on adult human pulmonary microvascular endothelial cells (ECs) after inhibiting Notch cleavage. Similar mechanisms were studied in PH mouse models and pulmonary arterial hypertension patients. The rescue effects of constitutively activated Notch1 in vivo were also measured. We observed that Dll4nAbs induced PH in mice as indicated by significantly increased right ventricular systolic pressure, as well as pulmonary vascular and right ventricular remodeling. Mechanistically, Dll4nAbs inhibited Notch1 cleavage and subsequently impaired lung endothelial barrier function and increased immune cell infiltration in vessel walls. In vitro, Notch targeted genes' expression related to cell growth and inflammation was decreased in human pulmonary microvascular ECs after the Notch1 inactivation. In lungs of PH mouse models and pulmonary arterial hypertension patients, Notch1 cleavage was inhibited. Consistently, EC cell-cell junction was leaky, and immune cell infiltration increased in PH mouse models. Overexpression activated Notch1-attenuated progression of PH in mice. In conclusion, Dll4nAbs led to PH development in mice by impaired EC barrier function and increased immune cell infiltration through inhibition of Notch1 cleavage in lung ECs. Reduced Notch1 cleavage in lung ECs could be an underlying mechanism of PH pathogenesis.

Cyclic nucleotide phosphodiesterase 1C contributes to abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by aorta dilation due to wall degeneration, which mostly occurs in elderly males. Vascular aging is implicated in degenerative vascular pathologies, including AAA. Cyclic nucleotide phosphodiesterases, by hydrolyzing cyclic nucleotides, play critical roles in regulating vascular structure remodeling and function. Cyclic nucleotide phosphodiesterase 1C (PDE1C) expression is induced in dedifferentiated and aging vascular smooth muscle cells (SMCs), while little is known about the role of PDE1C in aneurysm. We observed that PDE1C was not expressed in normal aorta but highly induced in SMC-like cells in human and murine AAA. In mouse AAA models induced by Angiotensin II or periaortic elastase, PDE1C deficiency significantly decreased AAA incidence, aortic dilation, and elastin degradation, which supported a causative role of PDE1C in AAA development in vivo. Pharmacological inhibition of PDE1C also significantly suppressed preestablished AAA. We showed that PDE1C depletion antagonized SMC senescence in vitro and/or in vivo, as assessed by multiple senescence biomarkers, including senescence-associated ß-galactosidase activity, γ-H2AX foci number, and p21 protein level. Interestingly, the role of PDE1C in SMC senescence in vitro and in vivo was dependent on Sirtuin 1 (SIRT1). Mechanistic studies further showed that cAMP derived from PDE1C inhibition stimulated SIRT1 activation, likely through a direct interaction between cAMP and SIRT1, which leads to subsequent up-regulation of SIRT1 expression. Our findings provide evidence that PDE1C elevation links SMC senescence to AAA development in both experimental animal models and human AAA, suggesting therapeutical significance of PDE1C as a potential target against aortic aneurysms.

Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin.

[Figure: see text].

MicroRNA-574 regulates FAM210A expression and influences pathological cardiac remodeling.

Aberrant expression of mitochondrial proteins impairs cardiac function and causes heart disease. The mechanism of regulation of mitochondria encoded protein expression during cardiac disease, however, remains underexplored. Here, we show that multiple pathogenic cardiac stressors induce the expression of miR-574 guide and passenger strands (miR-574-5p/3p) in both humans and mice. miR-574 knockout mice exhibit severe cardiac disorder under different pathogenic cardiac stresses while miR-574-5p/3p mimics that are delivered systematically using nanoparticles reduce cardiac pathogenesis under disease insults. Transcriptomic analysis of miR-574-null hearts uncovers family with sequence similarity 210 member A (FAM210A) as a common target mRNA of miR-574-5p and miR-574-3p. The interactome capture analysis suggests that FAM210A interacts with mitochondrial translation elongation factor EF-Tu. Manipulating miR-574-5p/3p or FAM210A expression changes the protein expression of mitochondrial-encoded electron transport chain (ETC) genes but not nuclear-encoded mitochondrial ETC genes in both human AC16 cardiomyocyte cells and miR-574-null murine hearts. Together, we discovered that miR-574 regulates FAM210A expression and modulates mitochondrial-encoded protein expression, which may influence cardiac remodeling in heart failure.

Sex-Specific Platelet Activation Through Protease-Activated Receptors Reverses in Myocardial Infarction.

OBJECTIVE: The platelet phenotype in certain patients and clinical contexts may differ from healthy conditions. We evaluated platelet activation through specific receptors in healthy men and women, comparing this to patients presenting with ST-segment-elevation myocardial infarction and non-ST-segment-elevation myocardial infarction. Approach and Results: We identified independent predictors of platelet activation through certain receptors and a murine MI model further explored these findings. Platelets from healthy women and female mice are more reactive through PARs (protease-activated receptors) compared with platelets from men and male mice. Multivariate regression analyses revealed male sex and non-ST-segment-elevation myocardial infarction as independent predictors of enhanced PAR1 activation in human platelets. Platelet PAR1 signaling decreased in women and increased in men during MI which was the opposite of what was observed during healthy conditions. Similarly, in mice, thrombin-mediated platelet activation was greater in healthy females compared with males, and lesser in females compared with males at the time of MI. CONCLUSIONS: Sex-specific signaling in platelets seems to be a cross-species phenomenon. The divergent platelet phenotype in males and females at the time of MI suggests a sex-specific antiplatelet drug regimen should be prospectively evaluated.

Vinpocetine protects against the development of experimental abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA), commonly occurring in the aged population, is a degenerative disease that dilate and weaken infrarenal aorta due to progressive degeneration of aortic wall integrity. Vinpocetine, a derivative of alkaloid vincamine, has long been used for cerebrovascular disorders and cognitive impairment in the aged population. Recent studies have indicated that vinpocetine antagonizes occlusive vascular disorders such as intimal hyperplasia and atherosclerosis. However, its role in vascular degenerative disease AAA remains unexplored. Herein, we determined the effect of vinpocetine on the formation of AAA as well as the intervention of pre-existing moderate AAA. AAA was induced by periaortic elastase application in C57BL/6J mice. Systemic vinpocetine treatment was applied daily via intraperitoneal injection. We showed that vinpocetine pre-treatment remarkably attenuated aneurysmal dilation assessed by diameter and volume. More importantly, vinpocetine also significantly suppressed the progression of pre-existing moderate AAA in a post-intervention model. Vinpocetine improved multiple cellular and molecular changes associated with AAA, such as elastin degradation, media smooth muscle cell depletion, collagen fibers remodeling and macrophage infiltration in aneurysmal tissues. Vinpocetine potently suppressed tumor necrosis factor-α-induced nuclear factor kappa-light-chain-enhancer of activated B cells activation and proinflammatory mediator expression in primary cultured macrophages in vitro, as well as in the aorta wall in vivo, suggesting vinpocetine conferred anti-AAA effect at least partially via the inhibition of inflammation. Taken together, our findings reveal a novel role of vinpocetine in AAA formation, development and progression. Given the excellent safety profile of vinpocetine, the present study suggests vinpocetine may be a novel therapeutic agent for AAA prevention and treatment.

Platelet-derived ß2m regulates age related monocyte/macrophage functions.

Platelets have central roles in both immune responses and development. Stimulated platelets express leukocyte adhesion molecules and release numerous immune modulatory factors that recruit and activate leukocytes, both at the sites of activation and distantly. Monocytes are innate immune cells with dynamic immune modulatory functions that change during the aging process, a phenomenon termed "inflammaging". We have previously shown that platelets are a major source of plasma beta-2 microglobulin (ß2M) and that ß2M induced a monocyte pro-inflammatory phenotype. Plasma ß2M increases with age and is a pro-aging factor. We hypothesized that platelet derived ß2M regulates monocyte phenotypes in the context of aging. Using wild-type (WT) and platelet specific ß2M knockout mice (Plt-ß2M-/-) mice, we found that plasma ß2M increased with age and correlated with increased circulating Ly6CHi monocytes. However, aged Plt-ß2M-/- mice had significantly fewer Ly6CHi monocytes compared to WT mice. Quantitative real-time PCR of circulating monocytes showed that WT mouse monocytes were more "pro-inflammatory" with age, while Plt-ß2M-/- derived monocytes adopted a "pro-reparative" phenotype. Older Plt-ß2M-/- mice had a significant decline in heart function compared to age matched WT mice, as well as increased cardiac fibrosis and pro-fibrotic markers. These data suggest that platelet-derived ß2M regulates age associated monocyte polarization, and a loss of platelet derived ß2M shifted monocytes and macrophages to a pro-reparative phenotype and increased pro-fibrotic cardiac responses. Platelet regulation of monocyte phenotypes via ß2M may maintain a balance between inflammatory and reparative signals that affects age related physiologic outcomes.

Platelet-derived ß2M regulates monocyte inflammatory responses.

ß-2 Microglobulin (ß2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but ß2M may also have less understood chaperone-independent functions. Elevated plasma ß2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. ß2M mRNA is present in platelets at very high levels, and ß2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived ß2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFß receptor signaling. Circulating monocytes from mice lacking ß2M only in platelets (Plt-ß2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFß signaling in the absence of ß2M. Using a mouse myocardial infarction (MI) model, Plt-ß2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet ß2M and that platelet-derived 2M and TGFß have opposing roles in monocyte differentiation that may be important in tissue injury responses.

Multiprotein Complex With TRPC (Transient Receptor Potential-Canonical) Channel, PDE1C (Phosphodiesterase 1C), and A2R (Adenosine A2 Receptor) Plays a Critical Role in Regulating Cardiomyocyte cAMP and Survival.

BACKGROUND: cAMP plays a critical role in regulating cardiomyocyte survival. Various cAMP signaling pathways behave distinctly or in opposition. We have previously reported that activation of cAMP hydrolysis by cyclic nucleotide phosphodiesterase 1C (PDE1C) promotes cardiomyocytes death/apoptosis, yet the underlying molecular mechanism remains unknown. In this study, we aimed to identify the specific cAMP signaling pathway modulated by PDE1C and determine the mechanism by which Ca2+/calmodulin-stimulated PDE1C is activated. METHODS: To study cardiomyocyte death/apoptosis, we used both isolated mouse adult cardiomyocytes in vitro and doxorubicin-induced cardiotoxicity in vivo. We used a variety of pharmacological activators and inhibitors as well as genetically engineered molecular tools to manipulate the expression and activity of proteins of interest. RESULTS: We found that the protective effect of PDE1C inhibition/deficiency on Ang II or doxorubicin-induced cardiomyocyte death/apoptosis is dependent on cAMP-generating adenosine A2 receptors (A2Rs), suggesting that PDE1C's cAMP-hydrolyzing activity selectively modulates A2R-cAMP signaling in cardiomyocytes. In addition, we found that the effects of PDE1C activation on Ang II-mediated cAMP reduction and cardiomyocyte death are dependent on transient receptor potential-canonical (TRPC) channels, in particular TRPC3. We also observed synergistic protective effects on cardiomyocyte survival from the combination of A2R stimulation together with PDE1 or TRPC inhibition. Coimmunostaining and coimmunoprecipitation studies showed that PDE1C is localized in proximity with A2R and TRPC3 in the plasma membrane and perhaps T tubules. It is important to note that we found that doxorubicin-induced cardiac toxicity and dysfunction in mice are attenuated by the PDE1 inhibitor IC86340 or in PDE1C knockout mice, and this protective effect is significantly diminished by A2R antagonism. CONCLUSIONS: We have characterized a novel multiprotein complex comprised of A2R, PDE1C, and TRPC3, in which PDE1C is activated by TRPC3-derived Ca2+, thereby antagonizing A2R-cAMP signaling and promoting cardiomyocyte death/apoptosis. Targeting these molecules individually or in combination may represent a compelling therapeutic strategy for potentiating cardiomyocyte survival.

Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype.

OBJECTIVE: Reduced blood flow and tissue oxygen tension conditions result from thrombotic and vascular diseases such as myocardial infarction, stroke, and peripheral vascular disease. It is largely assumed that while platelet activation is increased by an acute vascular event, chronic vascular inflammation, and ischemia, the platelet activation pathways and responses are not themselves changed by the disease process. We, therefore, sought to determine whether the platelet phenotype is altered by hypoxic and ischemic conditions. APPROACH AND RESULTS: In a cohort of patients with metabolic and peripheral artery disease, platelet activity was enhanced, and inhibition with oral antiplatelet agents was impaired compared with platelets from control subjects, suggesting a difference in platelet phenotype caused by the disease. Isolated murine and human platelets exposed to reduced oxygen (hypoxia chamber, 5% O2) had increased expression of some proteins that augment platelet activation compared with platelets in normoxic conditions (21% O2). Using a murine model of critical limb ischemia, platelet activity was increased even 2 weeks postsurgery compared with sham surgery mice. This effect was partly inhibited in platelet-specific ERK5 (extracellular regulated protein kinase 5) knockout mice. CONCLUSIONS: These findings suggest that ischemic disease changes the platelet phenotype and alters platelet agonist responses because of changes in the expression of signal transduction pathway proteins. Platelet phenotype and function should, therefore, be better characterized in ischemic and hypoxic diseases to understand the benefits and limitations of antiplatelet therapy.

G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation.

OBJECTIVE: Recent evidence suggests G-protein-coupled receptor-2-interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. APPROACH AND RESULTS: In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. CONCLUSION: Our data demonstrated that a GIT1-cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.

G protein coupled receptor kinase 2 interacting protein 1 (GIT1) is a novel regulator of mitochondrial biogenesis in heart.

G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-function scaffold protein. However, little is known about its physiological role in the heart. Here we sought to identify the cardiac function of GIT1. Global GIT1 knockout (KO) mice were generated and exhibited significant cardiac hypertrophy that progressed to heart failure. Electron microscopy revealed that the hearts of GIT1 KO mice demonstrated significant morphological abnormities in mitochondria, including decreased mitochondrial volume density, cristae density and increased vacuoles. Moreover, mitochondrial biogenesis-related gene peroxisome proliferator-activated receptor γ (PPARγ) co-activator-1α (PGC-1α), PGC-1ß, mitochondrial transcription factor A (Tfam) expression, and total mitochondrial DNA were remarkably decreased in hearts of GIT1 KO mice. These animals also had impaired mitochondrial function, as evidenced by reduced ATP production and dissipated mitochondrial membrane potential (Ψ(m)) in adult cardiomyocytes. Concordant with these mitochondrial observations, GIT1 KO mice showed enhanced cardiomyocyte apoptosis and cardiac dysfunction. In conclusion, our findings identify GIT1 as a new regulator of mitochondrial biogenesis and function, which is necessary for postnatal cardiac maturation.

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. CONCLUSIONS: 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.

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

Cyclophilin A (CyPA; encoded by Ppia) is a ubiquitously expressed protein secreted in response to inflammatory stimuli. CyPA stimulates vascular smooth muscle cell migration and proliferation, endothelial cell adhesion molecule expression, and inflammatory cell chemotaxis. Given these activities, we hypothesized that CyPA would promote atherosclerosis. Apolipoprotein E-deficient (Apoe(-/-)) mice fed a high-cholesterol diet for 16 wk developed more severe atherosclerosis compared with Apoe(-/-)Ppia(-/-) mice. Moreover, CyPA deficiency was associated with decreased low-density lipoprotein uptake, VCAM-1 (vascular cell adhesion molecule 1) expression, apoptosis, and increased eNOS (endothelial nitric oxide synthase) expression. To understand the vascular role of CyPA in atherosclerosis development, bone marrow (BM) cell transplantation was performed. Atherosclerosis was greater in Apoe(-/-) mice compared with Apoe(-/-)Ppia(-/-) mice after reconstitution with CyPA(+/+) BM cells, indicating that vascular-derived CyPA plays a crucial role in the progression of atherosclerosis. These data define a role for CyPA in atherosclerosis and suggest CyPA as a target for cardiovascular therapies.

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.

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 approximately 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-Cgamma (PLCgamma) 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 PLCgamma and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCgamma and ERK1/2. PLCgamma 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 PLCgamma and ERK1/2 activation.

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.