Cavin-2 loss exacerbates hypoxia-induced pulmonary hypertension with excessive eNOS phosphorylation and protein nitration.

Pulmonary hypertension (PH) is associated with a poor prognosis even in recent years. Caveolin-1 (CAV1), a caveolae-associated protein, is a causal gene in PH. Cavin-2, one of the other caveolae-associated proteins, forms protein complexes with CAV1 and influences each other's functions. However, the role of Cavin-2 in PH has not been thoroughly investigated. To clarify the role of Cavin-2 in PH, we exposed Cavin-2-deficient (Cavin-2 KO) mice to hypoxia. A part of the analyses was confirmed in human pulmonary endothelial cells (HPAECs). After 4-week 10% O2 hypoxic exposure, we performed physiological, histological, and immunoblotting analyses. Right ventricular (RV) systolic pressure elevation and RV hypertrophy were exacerbated in Cavin-2 KO mice with hypoxia-induced PH (Cavin-2 KO PH mice). The vascular wall thickness of pulmonary arterioles was aggravated in Cavin-2 KO PH mice. Cavin-2 loss reduced CAV1 and induced sustained endothelial nitric oxide synthase (eNOS) hyperphosphorylation in the Cavin-2 KO PH lungs and HPAECs. NOx production associated with eNOS phosphorylation was also increased in the Cavin-2 KO PH lung and HPAECs. Furthermore, the nitration of proteins, including protein kinase G (PKG), was raised in the Cavin-2 KO PH lungs. In conclusion, we revealed that Cavin-2 loss exacerbated hypoxia-induced PH. Our results suggest that Cavin-2 loss leads to sustained eNOS hyperphosphorylation in pulmonary artery endothelial cells via CAV1 reduction, resulting in Nox overproduction-mediated nitration of proteins, including PKG, in smooth muscle cells.

Clot-regression effects of rivaroxaban in venous thromboembolism treatment in cancer patients-a prospective interventional study.

Rivaroxaban, a direct oral anticoagulant, is effective against venous thromboembolism (VTE) recurrence without increasing the risk of major bleeding in patients with cancer-associated venous thromboembolism (CAT). However, its clot regression effects are poorly understood. This single-arm, prospective interventional study aimed to investigate the clot regression effects of rivaroxaban in 40 CAT patients, through a contrast-enhanced computed tomography at baseline, 3 weeks, and 3 months of rivaroxaban treatment. The primary endpoint was the clot-regression ratio calculated from the thrombus volumes at 3 weeks and 3 months. Compared with baseline, the total clot volume was significantly reduced at both 3 weeks and 3 months after initiation (p < 0.01). The clot-regression rates were statistically significant with 83.1% (95% confidence interval [CI], 73.8-92.3%) at 3 weeks and 98.7% (95% CI, 97.1-100.2%) at 3 months, with complete resolution in 36.1% and 80.8% of patients at 3 weeks and 3 months, respectively. One patient had recurrent VTE after dose reduction, and seven had non-fatal major bleeding. Therefore, rivaroxaban had a sufficient clot-regression effect against CAT with caution of bleeding complication.

Prognostic value of left ventricular mechanical dyssynchrony induced by exercise stress in patients with normal myocardial perfusion single-photon emission computed tomography.

BACKGROUND: Left ventricular mechanical dyssynchrony (LVMD) induced by exercise stress was reported to be clinically useful in detecting multivessel coronary artery diseases. The aim of this study was to compare the prognostic value of LVMD induced by pharmacological stress with that induced by exercise stress. METHODS: We retrospectively examined 918 consecutive patients who underwent exercise (N = 310) or pharmacological stress (N = 608) 99mTc-tetrofosmin single-photon emission computed tomography (SPECT) with normal myocardial perfusion. LVMD was evaluated by phase analysis as the indices of phase bandwidth and phase standard deviation (PSD). RESULTS: During the follow-up period (2.2 ± 1.9 years), 74 major cardiac events (MCEs) occurred (7 cases of cardiac death, 17 cases of heart failure, and 50 cases of coronary intervention). In global patients, the indices of LVMD on rest images were significantly greater in patients with MCEs (bandwidth (°): 51 ± 31 vs 37 ± 21, P = .001, PSD: 14 ± 9 vs 10 ± 6, P = .001). The exercise stress bandwidth was significantly higher in patients with MCEs (62 ± 37° vs 42 ± 21°, P = .026), as was the pharmacological stress bandwidth (57 ± 35° vs 43 ± 24°, P = .006). Multivariate analysis demonstrated the exercise stress bandwidth to be an independent predictor of MCEs (HR 1.017, CI 1.003 to 1.032, P = .019), but the pharmacological stress bandwidth had no influence on MCEs. CONCLUSIONS: LVMD induced by exercise stress was an independent predictor of MCEs in patients with normal perfusion SPECT, whereas that induced by pharmacological stress had no association with further events.

IVUS-Guided Wiring Improves the Clinical Outcomes of Angioplasty for Long Femoropopliteal CTO Compared with the Conventional Intraluminal Approach.

AIMS: This study aimed to assess the clinical efficacy of intravascular ultrasound (IVUS)-guided intraplaque wiring for femoropopliteal (FP) chronic total occlusion (CTO). METHODS: This single-center, retrospective, observational study was performed at the Japanese Red Cross Kyoto Daini Hospital. From March 2013 to June 2017, a total of 75 consecutive patients (mean age: 75.4±8.5 years; 59 males), who underwent endovascular treatment (EVT), having 82 de novo FP-CTO lesions, were enrolled in this study. Eleven of the lesions that met the exclusion criteria were excluded, and the remaining 71 lesions were divided into the IVUS-guided wiring group (n=34) and non-IVUS-guided wiring group (n=37). Primary patency, defined as a peak systolic velocity ratio of ＜2.4 on duplex ultrasonography, and freedom from clinically driven target lesion revascularization (CD-TLR) at 12 months were the primary outcomes. RESULTS: The mean lesion length was 21.6±8.9 cm. The frequencies of primary patency and freedom from CD-TLR were significantly higher in the IVUS-guided wiring group than in the non-IVUS-guided wiring group (70.0% vs. 52.2%, p=0.045; 83.9% vs. 62.8%, p=0.036, respectively). The complete clinically true lumen angioplasty rate was also higher in the IVUS-guided wiring group than in the non-IVUS-guided wiring group (91.1% vs. 51.3%, p＜0.001, respectively). The clinically true and false wire passage rates were respectively 97.3% and 2.7% in the IVUS-guided wiring group. CONCLUSION: IVUS-guided wiring improves the clinical outcomes of EVT for FP-CTO by achieving a high clinically true lumen wire passage rate.

The frequency and clinical characteristics of in-stent restenosis due to calcified nodule development after coronary stent implantation.

The purpose of this study was to evaluated the clinical characteristics of calcified nodule-like in-stent restenosis (ISR) lesions using optical coherence tomography (OCT) in vivo. A total of 124 ISR lesions that were treated with a repeat coronary intervention under OCT guidance were included in this analysis. ISR neointimal morphology was classified as "calcified nodule-like ISR", that appeared as a high-backscattering protruding mass with an irregular surface covered by signal-rich bands, or "non-calcified nodule-like ISR". The maximum arc and thickness of calcium behind the stent struts was also measured. Of the 124 ISR lesions, calcified nodule-like ISR was observed in 11 lesions (9%). OCT analysis data showed that the maximum arc of calcium and the maximum calcium thickness behind the stent were significantly larger in the calcified nodule-like ISR lesions than in the non-calcified nodule-like ISR lesions (269 ± 51 vs. 179 ± 92°, p < 0.01 and 989 ± 174 vs. 684 ± 241 µm, p < 0.01, respectively). The enlargement of the stent area was significantly larger in the calcified nodule-like ISR lesions than in the non-calcified nodule-like ISR lesions (1.6 ± 2.3 vs. 0.7 ± 1.3 mm2, p = 0.02). As a result, the enlargement of the lumen area tended to be larger in the calcified group (2.8 ± 1.7 vs. 2.4 ± 1.3 mm2, p = 0.3). Calcified nodule-like neointima within the stent could develop in approximately 10% of all ISR lesions, especially within stents deployed in severely calcified lesions.

Utility of angiography-physiology coregistration maps during percutaneous coronary intervention in clinical practice.

This study aimed to evaluate the utility and feasibility of physiological maps coregistered with angiograms using the pullback of a pressure guidewire with continuous instantaneous wave-free ratio (iFR) measurements. iFR pullback was obtained for 70 lesions from 70 patients with stable angina pectoris using SyncVision (Philips Corp.). Physiological maps were created, whereby the post-intervention iFR (post-iFR) was predicted as iFRpred. The iFR gap was defined if the difference between the iFRpred and post-iFR was ≥ 0.3. The lesion morphology changed from that during the physiological assessment to that during the angiographic assessment in 26 lesions (37.1%). In particular, 22.6% of angiographic tandem lesions changed to physiological focal lesions. The mean pre-intervention iFR, post-iFR, and iFRpred were 0.73 ± 0.17, 0.90 ± 0.06, and 0.93 ± 0.05, respectively. The mean difference between the iFRpred and post-iFR was 0.029 ± 0.099, with 95% limits of agreement of -0.070-0.128. iFR gaps occurred in 28 patients (40%). Notably, a new iFR gradient causing a ≥ 0.03 iFR drop after stenting occurred in 11 (15.7%) cases. The study patients were divided into two groups according to biases between post-iFR and iFRpred < 0.03 (good concordance group) or ≥ 0.03 (poor concordance group). The pre-intervention heart rate was the only independent predictor of poor concordance (odds ratio, 0.936; 95% confidence interval 0.883-0.992; p = 0.027). Physiological maps under resting conditions may contribute to a reduction in unnecessary stent placements without missing lesions requiring treatment. However, the predictive accuracy of post-iFR performance in the present study was slightly lower than that in the previous reports.

Loss of MURC/Cavin-4 induces JNK and MMP-9 activity enhancement in vascular smooth muscle cells and exacerbates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is relatively common in elderly patients with atherosclerosis. MURC (muscle-restricted coiled-coil protein)/Cavin-4 modulating the caveolae function of muscle cells is expressed in cardiomyocytes, skeletal muscle cells and smooth muscle cells. Here, we show a novel functional role of MURC/Cavin-4 in vascular smooth muscle cells (VSMCs) and AAA development. Both wild-type (WT) and MURC/Cavin-4 knockout (MURC-/-) mice subjected to periaortic application of CaCl2 developed AAAs. Six weeks after CaCl2 treatment, internal and external aortic diameters were significantly increased in MURC-/- AAAs compared with WT AAAs, which were accompanied by advanced fibrosis in the tunica media of MURC-/- AAAs. The activity of JNK and matrix metalloproteinase (MMP) -2 and -9 were increased in MURC-/- AAAs compared with WT AAAs at 5 days after CaCl2 treatment. At 6 weeks after CaCl2 treatment, MURC-/- AAAs exhibited attenuated JNK activity compared with WT AAAs. There was no difference in the activity of MMP-2 or -9 between saline and CaCl2 treatments. In MURC/Cavin-4-knockdown VSMCs, TNFα-induced activity of JNK and MMP-9 was enhanced compared with control VSMCs. Furthermore, WT, MURC-/-, apolipoprotein E-/- (ApoE-/-), and MURC/Cavin-4 and ApoE double-knockout (MURC-/-ApoE-/-) mice were subjected to angiotensin II (Ang II) infusion. In both ApoE-/- and MURC-/-ApoE-/- mice infused for 4 weeks with Ang II, AAAs were promoted. The internal aortic diameter was significantly increased in Ang II-infused MURC-/-ApoE-/- mice compared with Ang II-infused ApoE-/- mice. In MURC/Cavin-4-knockdown VSMCs, Ang II-induced activity of JNK and MMP-9 was enhanced compared with control VSMCs. Our results suggest that MURC/Cavin-4 in VSMCs modulates AAA progression at the early stage via the activation of JNK and MMP-9. MURC/Cavin-4 is a potential therapeutic target against AAA progression.

PTRF/Cavin-1 Deficiency Causes Cardiac Dysfunction Accompanied by Cardiomyocyte Hypertrophy and Cardiac Fibrosis.

Mutations in the PTRF/Cavin-1 gene cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. Additionally, long-QT syndrome and fatal cardiac arrhythmia are observed in patients with CGL4 who have homozygous PTRF/Cavin-1 mutations. PTRF/Cavin-1 deficiency shows reductions of caveolae and caveolin-3 (Cav3) protein expression in skeletal muscle, and Cav3 deficiency in the heart causes cardiac hypertrophy with loss of caveolae. However, it remains unknown how loss of PTRF/Cavin-1 affects cardiac morphology and function. Here, we present a characterization of the hearts of PTRF/Cavin-1-null (PTRF-/-) mice. Electron microscopy revealed the reduction of caveolae in cardiomyocytes of PTRF-/- mice. PTRF-/- mice at 16 weeks of age developed a progressive cardiomyopathic phenotype with wall thickening of left ventricles and reduced fractional shortening evaluated by echocardiography. Electrocardiography revealed that PTRF-/- mice at 24 weeks of age had low voltages and wide QRS complexes in limb leads. Histological analysis showed cardiomyocyte hypertrophy accompanied by progressive interstitial/perivascular fibrosis. Hypertrophy-related fetal gene expression was also induced in PTRF-/- hearts. Western blotting analysis and quantitative RT-PCR revealed that Cav3 expression was suppressed in PTRF-/- hearts compared with that in wild-type (WT) ones. ERK1/2 was activated in PTRF-/- hearts compared with that in WT ones. These results suggest that loss of PTRF/Cavin-1 protein expression is sufficient to induce a molecular program leading to cardiomyocyte hypertrophy and cardiomyopathy, which is partly attributable to Cav3 reduction in the heart.

MURC deficiency in smooth muscle attenuates pulmonary hypertension.

Emerging evidence suggests that caveolin-1 (Cav1) is associated with pulmonary arterial hypertension. MURC (also called Cavin-4) is a member of the cavin family, which regulates caveolar formation and functions together with caveolins. Here, we show that hypoxia increased Murc mRNA expression in the mouse lung, and that Murc-null mice exhibited attenuation of hypoxia-induced pulmonary hypertension (PH) accompanied by reduced ROCK activity in the lung. Conditional knockout mice lacking Murc in smooth muscle also resist hypoxia-induced PH. MURC regulates the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through Rho/ROCK signalling. Cav1 suppresses RhoA activity in PASMCs, which is reversed by MURC. MURC binds to Cav1 and inhibits the association of Cav1 with the active form of Gα13, resulting in the facilitated association of the active form of Gα13 with p115RhoGEF. These results reveal that MURC has a function in the development of PH through modulating Rho/ROCK signalling.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.