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Background: Exposure to chronic psychological stress is a risk factor for metabolic cardiovascular disease. Given the important role of lysosomal CTSS (cathepsin S) in human pathobiology, we examined the role of CTSS in stress-related thrombosis, focusing on inflammation, oxidative stress, and apoptosis., Methods: Six-week-old wild-type mice (CTSS +/+ ) and CTSS-deficient mice (CTSS -/- ) randomly assigned to nonstress and 2-week immobilization stress groups underwent iron chloride3 (FeCl 3 )-induced carotid thrombosis surgery for morphological and biochemical studies., Results: On day 14 poststress/surgery, stress had increased the lengths and weights of thrombi in the CTSS +/+ mice, plus harmful changes in the levels of PAI-1 (plasminogen activation inhibitor-1), ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 13 motifs), and vWF (von Willebrand factor) and arterial tissue CTSS expression. Compared to the nonstressed CTSS +/+ mice, the stressed CTSS -/- mice had decreased levels of PAI-1, vWF, TNF (tumor necrosis factor)-α, interleukin-1β, toll-like receptor-4, cleaved-caspase 3, cytochrome c , p16 INK4A , gp91 phox , p22 phox , ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein-1), MyD88 (myeloid differentiation primary response 88), and MMP (matrix metalloproteinase)-2/-9 and increased levels of ADAMTS13, SOD (superoxide dismutase)-1/-2, eNOS (endothelial NO synthase), p-Akt (phospho-protein kinase B), Bcl-2 (B-cell lymphoma-2), p-GSK3α/β (phospho-glycogen synthase kinases alpha and beta), and p-Erk1/2 (phospho-extracellular signal-regulated kinase 1 and 2) mRNAs and/or proteins. CTSS deletion also reduced the arterial thrombus area and endothelial loss. A pharmacological inhibition of CTSS exerted a vasculoprotective action. In vitro, CTSS silencing and overexpression, respectively, reduced and increased the stressed serum and oxidative stress-induced apoptosis of human umbilical vein endothelial cells, and they altered apoptosis-related proteins., Conclusions: CTSS inhibition appeared to improve the stress-related thrombosis in mice that underwent FeCl 3 -induction surgery, possibly by reducing vascular inflammation, oxidative stress, and apoptosis. CTSS could thus become a candidate therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disease., Competing Interests: Disclosures None.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune demyelinating disease of the central nervous system. Although recent reports have noted that cognitive impairment is common in NMOSD, little longitudinal information is available on the trajectories of cognitive function in the disease. Here, we report a case of a 55-year-old woman with an 11-year history of NMOSD who visited our memory clinic for progressive memory loss. She was diagnosed with early-onset Alzheimer disease based on amyloid and tau positron emission tomography imaging biomarkers. This is the first report of early-onset Alzheimer disease in a patient with NMOSD. Complications of Alzheimer disease should be considered when patients with NMOSD exhibit rapid cognitive decline. More longitudinal studies of NMOSD with cognitive impairment are needed., Competing Interests: The authors declare no conflicts of interest.

Objective: Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice., Approach and Results: Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor-induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation., Conclusions: This is the first report detailing cross-interaction between toll-like receptor 2-mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation., (© 2016 The Authors.)

Objective: To investigate the mechanism responsible for the increased cardiac stiffness associated with hypertensive heart failure in Dahl salt-sensitive (DS) rats and the effects of treatment with the combination of a calcium channel blocker [azelnidipine (AZE)] and angiotensin II type 1 receptor blocker [olmesartan (OLM)]., Methods: DS rats fed a high-salt diet from 7 weeks of age were treated (or not) from 12 to 19 weeks of age with the vasodilator hydralazine, OLM plus AZE, or the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. Rats fed a low-salt diet served as controls., Results: Treatment with OLM plus AZE attenuated changes in the expression of collagen isoforms and a decrease in the ratio of elastin to collagen in the left ventricle and prevented the increase in myocardial stiffness and diastolic dysfunction in DS rats in a manner independent of the hypotensive effect of these drugs. Such treatment also inhibited the expression and activation of elastolytic proteases (including cathepsins S and K and metalloproteinases-2, -9, and -12), NADPH oxidase-dependent superoxide production, and inflammatory changes in the failing myocardium. All these effects were mimicked by treatment with apocynin., Conclusions: The changes in collagen isoform expression and the decrease in the elastin to collagen ratio in the failing myocardium likely account for the increase in diastolic stiffness in this model of hypertensive heart failure. Administration of angiotensin receptor and calcium channel blockers prevented these changes in a manner independent of the hypotensive effect of these drugs by inhibiting the increase in elastolytic activity induced by activation of NADPH oxidase.

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2(+/+) and MMP-2(-/-) mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2(-/-) young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti-l-lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2(-/-) mice compared with the age-matched MMP-2(+/+) mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2(-/-) mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow-derived endothelial progenitor cell (EPC)-like c-Kit(+) cells from MMP-2(-/-) showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2(-/-). Flow cytometry showed that the numbers of EPC-like CD31(+)c-Kit(+) cells in peripheral blood markedly decreased in MMP-2-deficient mice. Transplantation of bone marrow-derived mononuclear cells from MMP-2(+/+) mice restored neovascularization in MMP-2(-/-) young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.

Objective: The development of a murine model of atherosclerotic plaque rupture., Methods and Results: The left common carotid arteries of male apolipoprotein E (apoE)-deficient mice (9 weeks old) were ligated just proximal to their bifurcations. After 4 weeks on a standard diet, the mice received polyethylene cuff placement just proximal to the ligated site, and the animals were then processed for morphological studies at specific time points. Ligation of the carotid artery in apoE-deficient mice for 4 weeks induced marked intimal hyperplasia, which is a lipid- and collagen-rich lesion that contains a number of macrophages, T lymphocytes, and smooth muscle cells. Subsequently, the cuff placement evoked intraplaque hemorrhage and plaque rupture with fibrin(ogen)-positive luminal thrombus in this region accompanying a decrease in collagen content as well as an increase in apoptotic cells in the intima within a few days after cuff placement., Conclusions: We demonstrated the murine model of human plaque rupture, which is simple, fast, and highly efficient. This model would help us not only to understand the mechanism of human plaque rupture but also to assess various already-known and as-yet-unknown agents in the future.

Objective: Although it has been reported that matrix metalloproteinase (MMP)-2 is a major proteinase in atherosclerotic plaque lesions, there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using apolipoprotein E-deficient (apoE(-/-)) mice., Methods and Results: To generate MMP-2-deficient, apoE-deficient mice (MMP-2(-/-):apoE(-/-)), MMP-2(-/-) mice were crossed with apoE(-/-) mice. After 8 weeks of feeding with a lipid-rich diet, morphological and biochemical studies of the aortic sinus and arch were conducted. A significant reduction of the atherosclerotic plaque in the aortic sinus and arch with the decrease in smooth muscle cell-positive area was observed in MMP-2(-/-):apoE(-/-) mice compared with that of MMP-2(+/+):apoE(-/-) mice. Macrophage- and collagen-positive areas were less in aortic sinus but not in aortic arch in MMP-2(-/-):apoE(-/-) mice. There was no difference of MMP-9 mRNA expression in the plaque lesion between the 2 genotypes. A much lower level of mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesions of MMP-2(-/-):apoE(-/-) mice than in those of MMP-2(+/+):apoE(-/-) mice., Conclusions: MMP-2 contributes to the development of atherosclerosis in apoE(-/-) mice.

Objective: Although we recently showed that the administration of catechins reduced the neointimal formation in a rat balloon-injury model, the precise molecular mechanisms are largely unknown. In the present study, we tried to determine these mechanisms using an in vitro SMC invasion system., Methods and Results: Boyden chamber assay was used to examine the effect of catechins on the invasive behavior of SMCs. The invasive activity of SMCs through collagen gel was restrained by EGCG in a concentration-dependent manner. The data from gelatin and collagen zymography and Western blot revealed that EGCG blocks the activation of pro-matrix metalloproteinase (MMP)-2 during an invasion assay and in the conditioned medium of cultured SMCs as well as the activities of MMP-2 and membrane type 1-MMP (MT1-MMP) even at 0.1 to 0.3 micromol/L of EGCG. EGCG was found to restrain MT1-MMPcat-dependent pro-MMP-2 activation. EGCG upregulated the expression of tissue inhibitor of MMP-2 (TIMP-2) protein. Reverse zymography showed that the increased TIMP-2 to expression was validated by an increased activity. The data from decreased TIMP-2 activity using its siRNA suggested that upregulation of TIMP-2 expression may be one of the major mechanisms for inhibition of SMC invasion by EGCG., Conclusions: These results indicate that EGCG targets multiple MMP-mediated SMC cellular events and provides a new major mechanism for the SMC invasion through upregulation of TIMP-2 expression to modulate MMP activity.

Constrictive arterial remodeling plays a major role in lumen narrowing following angioplasty. We investigated the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, on vascular smooth muscle cell (SMC)-populated collagen lattice contraction, an in vitro model of vascular contraction. Type I collagen gel contraction by SMCs, which are cultured in collagen gel, was used as a model of vascular remodeling. Pitavastatin pretreatment inhibited 10% serum- or platelet-derived growth factor-BB (PDGF)-induced SMC-mediated collagen lattice contraction in a concentration-dependent manner. The effect of pitavastatin was prevented by mevalonate or geranylgeranyl pyrophosphate, but not by squalene, a precursor of cholesterol, or farnesyl pyrophosphate. The serum- or PDGF-induced SMC-mediated collagen gel contraction was inhibited by GGTI-298, a geranylgeranyltransferase inhibitor, C3 exoenzyme, an inhibitor of Rho, or Y27634, a Rho kinase inhibitor, but not by FTI-277, a farnesyltransferase inhibitor. Serum or PDGF treatment increased the stress fiber organization in SMCs, which was blocked by the pitavastatin pretreatment. Pitavastatin had no effect on the serum- and PDGF-induced lamelliopodia extension of SMC. These results may suggest that pitavastatin attenuates SMC-mediated collagen gel contraction probably via an inhibition of geranylgeranylated Rho protein and a disruption of actin cytoskeletal reorganization.

Background: Although it has been demonstrated that matrix metalloproteinases (MMPs) play an important role in the arterial remodeling in atherosclerosis and restenosis, it is not clear which MMP is involved in which process. To define the role of MMP-2 in arterial remodeling, we evaluated the influence of the targeted deletion of the MMP-2 gene on vascular remodeling after flow cessation in the murine carotid arteries., Methods and Results: The left common carotid arteries of wild-type and MMP-2-deficient mice were ligated just proximal to their bifurcations, and the animals were then processed for morphological and biochemical studies at specific time points. MMP-2 activity and mRNA levels increased in ligated carotid arteries of wild-type mice on the basis of observation by gelatin zymography and quantitative real-time RT-PCR. There was significantly less intimal hyperplasia in MMP-2-deficient mice at 2 and 4 weeks after ligation than there in wild-type mice. Arterial explants from the aorta of MMP-2-deficient mice showed that smooth muscle cell (SMC) migration was inhibited in comparison with wild-type mice. The chemoattractant-directed invasion through a reconstituted basement membrane barrier was significantly reduced in cultured SMCs derived from MMP-2-deficient mice, although no difference was observed in SMC migration across the filter or in proliferative response between the control and MMP-2-deficient mice., Conclusions: In a mouse carotid artery blood flow cessation model, MMP-2 contributes to intimal hyperplasia mainly through the SMC migration from the media into the intima by degrading and breaching the extracellular matrix proteins surrounding each cell and the internal elastic lamina.

Although the accumulation of vascular endothelial growth factor (VEGF) has been observed in human atherosclerotic lesions, the exact role of this growth factor in atherogenesis remains unknown. We hypothesized that VEGF in the vascular wall might have a preventive effect on endothelial cell damage during atherosclerosis. To test our hypothesis, we examined whether VEGF protects against the toxicity of oxidized low density lipoprotein (Ox-LDL) in cultured endothelial cells derived from bovine aortas (BAECs). Preincubation of BAECs with VEGF prevented Ox-LDL-induced toxicity in a preincubation time- and VEGF concentration-dependent manner. Addition of N(omega)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not reverse the protective effect of VEGF on Ox-LDL toxicity. Incubation of BAECs with VEGF increased intracellular glutathione (GSH) content in a time-dependent manner. Combined addition of VEGF and L-buthionine sulfoximine, a GSH synthesis inhibitor, reversed both GSH levels and the protective effect of VEGF on Ox-LDL-induced cytotoxicity. Placenta growth factor, which ligates to the VEGF Flt-1 receptor but not KDR/Flk-1, failed to prevent Ox-LDL toxicity and had no effect on intracellular GSH levels. An anti-KDR antibody completely blocked these beneficial activities of VEGF. These results suggest that VEGF prevents Ox-LDL-induced endothelial cell damage via an intracellular GSH-dependent mechanism through the KDR/Flk-1 receptor.

Smooth muscle cell (SMC) migration from the tunica media to the intima is a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. SMCs require not only migratory but also degradative abilities that enable them to migrate through extracellular matrix proteins, which surround and embed these cells. We used a collagen type I lattice as a coating on top of a porous filter as a matrix barrier in a chamber to test the invasive behavior of SMCs in response to a chemoattractant (invasion assay) and compared that behavior with simple SMC migration through collagen type I-coated filters (migration assay). Inhibitors of matrix metalloproteinase, KB-R8301, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TIMP-2, and peptide 74, attenuated platelet-derived growth factor-BB (PDGF-BB)-directed SMC invasion across the collagen lattice, whereas no effect was seen with these inhibitors on simple SMC migration through collagen-coated filters. RGD peptide inhibited SMC invasion but did not affect SMC migration. Anti-alphavbeta3 integrin antibody attenuated PDGF-BB-directed SMC invasion, whereas other antibodies against RGD-recognizing integrins, namely alphavbeta5 and alpha5, had no effect. None of these antibodies had any effect on simple SMC migration. RGD peptide and anti-alphavbeta3 antibody inhibited the attachment and spreading of SMCs on denatured collagen but not on native collagen. These findings indicate that there is a difference in the mechanisms between simple SMC migration across a collagen-coated filter and SMC invasion through a fibrillar collagen barrier. A proteolytic process is required for SMC invasion, and the degradation of matrix proteins alters the relationship between matrix protein molecules and SMC surface integrins.

The interaction between macrophages and oxidatively modified low density lipoprotein (Ox-LDL) appears to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the induction of numerous cytokines and growth factors. The current study demonstrated that Ox-LDL upregulated vascular endothelial growth factor (VEGF) mRNA expression in RAW 264 cells, a monocytic cell line, in a time- and concentration-dependent manner and that Ox-LDL stimulated VEGF protein secretion from the cells. Lysophosphatidylcholine, a component of Ox-LDL, also enhanced VEGF mRNA expression in RAW 264 cells and VEGF secretion from RAW 264 cells, with a maximal effect at a concentration of 10 micromol/L lysophosphatidylcholine. Immunohistochemical studies showed that human early atherosclerotic lesions exhibited intense VEGF immunoreactivity in subendothelial macrophage-rich regions of the thickened intima. In atherosclerotic plaques, VEGF staining was also observed in foam cell-rich regions adjacent to the lipid core or the neovascularized basal regions of plaque consisting predominantly of smooth muscle cells. High-power-field observation revealed that VEGF was localized in the extracellular space as well as at the macrophage cell surface. These observations suggest the possible involvement of Ox-LDL in the development of human atherosclerosis through VEGF induction in macrophages.