Formation of the atherosclerotic intima must involve altered metabolism of the elastin-rich arterial extracellular matrix. Proteases potentially involved in these processes remain unclear. This study examined the expression of the potent elastases cathepsins S and K in human atheroma. Normal arteries contained little or no cathepsin K or S. In contrast, macrophages in atheroma contained abundant immunoreactive cathepsins K and S. Intimal smooth muscle cells (SMC), especially cells appearing to traverse the internal elastic laminae, also contained these enzymes. Extracts of atheromatous tissues had approximately twofold greater elastase-specific activity than extracts of uninvolved arteries, mostly due to cysteine proteases.

Human atherosclerotic lesions overexpress the lysosomal cysteine protease cathepsin S (Cat S), one of the most potent mammalian elastases known. In contrast, atheromata have low levels of the endogenous Cat S inhibitor cystatin C compared with normal arteries, suggesting involvement of this protease in atherogenesis. The present study tested this hypothesis directly by crossing Cat S-deficient (CatS -/-) mice with LDL receptor-deficient (LDLR -/-) mice that develop atherosclerosis on a high-cholesterol diet. Compared with LDLR -/-mice, double-knockout mice (CatS -/-LDLR -/-) developed significantly less atherosclerosis, as indicated by plaque size (plaque area and intimal thickening) and stage of development. These mice also had markedly reduced content of intimal macrophages, lipids, smooth muscle cells, collagen, CD4 + T lymphocytes, and levels of IFN-γ. CatS -/-LDLR -/-monocytes showed impaired subendothelial basement membrane transmigration, and aortas from CatS -/-LDLR -/-mice had preserved elastic laminae. These findings establish a pivotal role for Cat S in atherogenesis.

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Objective This study tested whether IL-17A is involved in the pathogenesis of mouse myocardial ischemia-reperfusion (I/R) injury and investigated the mechanisms. Background Inflammatory processes play a major role in myocardial I/R injury. We recently identified interleukin (IL)-17A as an important cytokine in inflammatory cardiovascular diseases such as atherosclerosis and viral myocarditis. However, its role in myocardial I/R injury remains unknown. Methods The involvement of IL-17A was assessed in functional assays in mouse myocardial I/R injury by neutralization/repletion or genetically deficiency of IL-17A, and its mechanism on cardiomyocyte apoptosis and neutrophil infiltration were further studied in vivo and in vitro. Results IL-17A was elevated following murine left coronary artery ligation and reperfusion. Intracellular cytokine staining revealed that γδT lymphocytes, but not CD4+ helper T cells, were a major source of IL-17A. Anti-IL-17A mAb treatment or IL-17A knockout markedly ameliorated I/R injury, as demonstrated by reduced infarct size, reduced cardiac troponin T levels and improved cardiac function. This improvement was associated with a reduction in cardiomyocyte apoptosis and neutrophil infiltration. On the contrary, repletion of exogenous IL-17A induced the opposite effect. In vitro study showed that IL-17A mediated cardiomyocyte apoptosis through regulating the Bax/Bcl-2 ratio, induced CXC chemokine-mediated neutrophil migration and promoted neutrophil-endothelial cell adherence through induction of endothelial cell E-selectin and inter-cellular adhesion molecule (ICAM)-1 expression. Conclusions IL-17A mainly produced by γδT cells plays a pathogenic role in myocardial I/R injury by inducing cardiomyocyte apoptosis and neutrophil infiltration.

The cysteine protease cathepsin S is highly expressed in malignant tissues. By using a mouse model of multistage murine pancreatic islet cell carcinogenesis in which cysteine cathepsin activity has been functionally implicated, we demonstrated that selective cathepsin S deficiency impaired angiogenesis and tumor cell proliferation, thereby impairing angiogenic islet formation and the growth of solid tumors, whereas the absence of its endogenous inhibitor cystatin C resulted in opposite phenotypes. Although mitogenic vascular endothelial growth factor, transforming growth factor-␤1, and the anti-angiogenic endostatin levels in either serum or carcinoma tissue extracts did not change in cathepsin S-or cystatin C-null mice, tumor tissue basic fibroblast growth factor and serum type 1 insulin growth factor levels were higher in cystatin C-null mice, and serum type 1 insulin growth factor levels were also increased in cathepsin S-null mice. Furthermore, cathepsin S affected the production of type IV collagen-derived anti-angiogenic peptides and the generation of bioactive pro-angiogenic ␥2 fragments from laminin-5, revealing a functional role for cathepsin S in angiogenesis and neoplastic progression.Angiogenesis, the development of the microvasculature, is an essential process occurring under many pathological and physiological circumstances and depends on tightly controlled interactions between cells and extracellular matrix (ECM) 2 mediated by integral membrane proteins. These include integrins, which provide a link between ECM and the cytoskeleton, and extracellular proteases and their inhibitors, which mediate focal degradation of ECM components (1, 2), generate cell growth factors (3), and produce angiogenic regulatory factors (4).Lysosomal cysteine protease cathepsins have been shown to be highly expressed in human and murine tumors (5), where angiogenesis plays essential roles. Interruption of their expression either by antisense RNA (6) or RNA interference (7, 8) reduced tumor cell invasion, angiogenesis, and tumor growth. A recent study also demonstrated that inhibition of the activities of cysteine proteases with a broad inhibitor reduced angiogenesis and growth of pancreatic ␤-cell islet carcinoma in mice with an SV40 T antigen (Tag) transgene driven by the rat insulin II promoter (RIP1-Tag2) (9). Administration of JPM-ethyl ester (10), which affects all activities of cysteinyl cathepsins, significantly diminished the angiogenic switch, tumor burden, and tumor cell proliferation (11). However, many important questions are still unanswered; for example, which cathepsin(s) is the most important and by what mechanisms do these cysteinyl cathepsins affect tumor progression? Earlier studies suggested the importance of cathepsin (Cat) B in tumor angiogenesis and growth (6 -8). However, additional studies also demonstrated constitutive expression of Cat B in several cell types or tissues (12, 13). Therefore, cathepsins other than Cat B may also be involved in angiogenesis, tumor growth, cell proliferation, and...

During angiogenesis, microvascular endothelial cells (ECs) secrete proteinases that permit penetration of the vascular basement membrane as well as the interstitial extracellular matrix. This study tested the hypothesis that cathepsin S (Cat S) contributes to angiogenesis. Treatment of cultured ECs with inflammatory cytokines or angiogenic factors stimulated the expression of Cat S, whereas inhibition of Cat S activity reduced microtubule formation by impairing cell invasion. ECs from Cat S-deficient mice showed reduced collagenolytic activity and impaired invasion of collagens type I and IV. Cat S-deficient mice displayed defective microvessel development during wound repair. This abnormal angiogenesis occurred despite normal vascular endothelial growth factor and basic fibroblast growth factor levels, implying an essential role for extracellular matrix degradation by Cat S during microvessel formation. These results demonstrate a novel function of endothelium-derived Cat S in angiogenesis.

IgE has a key role in the pathogenesis of allergic responses through its ability to activate mast cells via the receptor FcεR1. In addition to mast cells, many cell types implicated in atherogenesis express FcεR1, but whether IgE has a role in this disease has not been determined. Here, we demonstrate that serum IgE levels are elevated in patients with myocardial infarction or unstable angina pectoris. We found that IgE and the FcεR1 subunit FcεR1α were present in human atherosclerotic lesions and that they localized particularly to macrophagerich areas. In mice, absence of FcεR1α reduced inflammation and apoptosis in atherosclerotic plaques and reduced the burden of disease. In cultured macrophages, the presence of TLR4 was required for FcεR1 activity. IgE stimulated the interaction between FcεR1 and TLR4, thereby inducing macrophage signal transduction, inflammatory molecule expression, and apoptosis. These IgE activities were reduced in the absence of FcεR1 or TLR4. Furthermore, IgE activated macrophages by enhancing Na + /H + exchanger 1 (NHE1) activity. Inactivation of NHE1 blocked IgE-induced macrophage production of inflammatory molecules and apoptosis. Cultured human aortic SMCs (HuSMCs) and ECs also exhibited IgE-induced signal transduction, cytokine expression, and apoptosis. In human atherosclerotic lesions, SMCs and ECs colocalized with IgE and TUNEL staining. This study reveals what we believe to be several previously unrecognized IgE activities that affect arterial cell biology and likely other IgE-associated pathologies in human diseases.

Background The molecular mechanisms that determine the localized formation of thin-capped atheromata in the coronary arteries remain unknown. This study tested the hypothesis that low endothelial shear stress augments the expression of matrix-degrading proteases and thereby promotes the formation of thin-capped atheromata. Methods and Results Intravascular ultrasound–based, geometrically correct 3-dimensional reconstruction of the coronary arteries of 12 swine was performed in vivo 23 weeks after initiation of diabetes mellitus and a hyperlipidemic diet. Local endothelial shear stress was calculated in plaque-free subsegments of interest (n=142) with computational fluid dynamics. At week 30, the coronary arteries (n=31) were harvested and the same subsegments were identified. The messenger RNA and protein expression and elastolytic activity of selected elastases and their endogenous inhibitors were assessed. Subsegments with low preceding endothelial shear stress at week 23 showed reduced endothelial coverage, enhanced lipid accumulation, and intense infiltration of activated inflammatory cells at week 30. These lesions showed increased expression of messenger RNAs encoding matrix metalloproteinase-2, -9, and -12, and cathepsins K and S relative to their endogenous inhibitors and increased elastolytic activity. Expression of these enzymes correlated positively with the severity of internal elastic lamina fragmentation. Thin-capped atheromata developed in regions with lower preceding endothelial shear stress and had reduced endothelial coverage, intense lipid and inflammatory cell accumulation, enhanced messenger RNA expression and elastolytic activity of MMPs and cathepsins, and severe internal elastic lamina fragmentation. Conclusions Low endothelial shear stress induces endothelial discontinuity and accumulation of activated inflammatory cells, thereby augmenting the expression and activity of elastases in the intima and shifting the balance with their inhibitors toward matrix breakdown. Our results provide new insight into the mechanisms of regional formation of plaques with thin fibrous caps.