Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Metabolic-associated fatty liver disease and the risk of cardiovascular disease.

BACKGROUND: With the gradual adoption of new metabolic-associated fatty liver disease (MAFLD) definitions in clinical practice, the relationship between MAFLD and cardiovascular disease (CVD) risk remains unclear. Similarly, clinical differences between MAFLD and nonalcoholic fatty liver disease (NAFLD), and the relationship between MAFLD and CVD risk are unclear. METHODS: We conducted a retrospective study using the 1988-1994 National Health and Nutrition Examination Surveys (NHANES III) database, including 11,673 individuals. Multivariate logistic regression analysis was performed to test relationships between MAFLD and the 10-year CVD risk. RESULTS: MAFLD was more significant than NAFLD in medium/high 10-year CVD risk (according to Framingham risk score) (1064 (29.92%) vs. 1022 (26.37%), P < 0.005). MAFLD patients were stratified according to NAFLD fibrosis scores (NFS's). In univariate regression analysis, when compared with non-MAFLD patients, unadjusted-OR values for MAFLD with different liver fibrosis stages, which were tiered by NFS (NFS < -1.455,-1.455 ≤ NFS < 0.676, and NFS ≥ 0.676) in the medium 10-year CVD risk (according to Framingham scores) were 1.175 (95% CI 1.030-1.341), 3.961 (3.449-4.549), and 5.477 (4.100-7.315), and the unadjusted or values of different MAFLD groups in the high 10-year CVD risk were 1.407 (95% CI 1.080-1.833), 5.725 (4.500-7.284), and 5.330 (3.132-9.068). Then, after adjusting for age, sex, race, alcohol consumption, and smoking, or adjusting for age, race, alcohol consumption, smoking, type 2 diabetes mellitus (T2DM), and other confounding factors, the incidence of medium and high 10-year CVD risk was statistically significant (P < 0.05). CONCLUSIONS: We showed that patients with MAFLD had a higher 10-year CVD risk when compared with patients with NAFLD. Increased MAFLD hepatic fibrosis scores were associated with a 10-year CVD risk.

Adiponectin suppresses angiotensin II-induced inflammation and cardiac fibrosis through activation of macrophage autophagy.

Previous studies have indicated that adiponectin (APN) protects against cardiac remodeling, but the underlying mechanism remains unclear. The present study aimed to elucidate how APN regulates inflammatory responses and cardiac fibrosis in response to angiotensin II (Ang II). Male APN knockout (APN KO) mice and wild-type (WT) C57BL/6 littermates were sc infused with Ang II at 750 ng/kg per minute. Seven days after Ang II infusion, both APN KO and WT mice developed equally high blood pressure levels. However, APN KO mice developed more severe cardiac fibrosis and inflammation compared with WT mice. This finding was demonstrated by the up-regulation of collagen I, α-smooth muscle actin, IL-1ß, and TNF-α and increased macrophage infiltration in APN KO mice. Moreover, there were substantially fewer microtubule-associated protein 1 light chain 3-positive autophagosomes in macrophages in the hearts of Ang II-infused APN KO mice. Additional in vitro studies also revealed that globular APN treatment induced autophagy, inhibited Ang II-induced nuclear factor-κB activity, and enhanced the expression of antiinflammatory cytokines, including IL-10, macrophage galactose N-acetyl-galactosamine specific lectin 2, found in inflammatory zone 1, and type-1 arginase in macrophages. In contrast, APN-induced autophagy and antiinflammatory cytokine expression was diminished in Atg5-knockdown macrophages or by Compound C, an inhibitor of adenosine 5'-monophosphate-activated protein kinase. Our study indicates that APN activates macrophage autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway and suppresses Ang II-induced inflammatory responses, thereby reducing the extent of cardiac fibrosis.

The requirement of CD8+ T cells to initiate and augment acute cardiac inflammatory response to high blood pressure.

Macrophage infiltration and activation in myocardium are hallmarks of acute cardiac inflammatory response to high blood pressure. However, the underlying mechanisms remain elusive. In this article, we report that CD8(+) T cells are required for cardiac recruitment and activation of macrophages. First, mice with CD8 gene-targeted (CD8 knockout) or CD8(+) T cells depleted by Ab showed significantly reduced cardiac inflammatory response to the elevation of blood pressure after angiotensin II (Ang II) infusion, whereas CD8 knockout mice reconstituted with CD8(+) T cells restored the sensitivity to Ang II. More importantly, CD8(+) T cells were required for macrophage infiltration in myocardium and subsequent activation to express proinflammatory cytokines and chemokines. Furthermore, macrophage activation required direct contact with activated CD8(+) T cells, but with TCR dispensable. TCR-independent activation of macrophages was further confirmed in MHC class I-restricted OVA-specific TCR transgenic mice, which showed a CD8(+) T cell activation and cardiac proinflammatory response to Ang II similar to that of wild-type mice. Finally, only myocardium-infiltrated, but not peripheral, CD8(+) T cells were specifically activated by Ang II, possibly by the cardiac IFN-γ that drove IFN-γR(+) CD8(+) T cell infiltration and activation. Thus, this work identified a TCR-independent innate nature of CD8(+) T cells that was critical in initiating the sterile immune response to acute elevation of blood pressure.

Inhibition of platelet activation by clopidogrel prevents hypertension-induced cardiac inflammation and fibrosis.

PURPOSE: Platelets are essential for primary hemostasis; however, platelet activation also plays an important proinflammatory role. Inflammation promotes the development of cardiac fibrosis and heart failure induced by hypertension. In this study, we aimed to determine whether inhibiting platelet activation using clopidogrel could inhibit hypertension-induced cardiac inflammation and fibrosis. METHODS: Using a mouse model of angiotensin II (Ang II) infusion (1,500 ng/[kg·min] for 7 days), we determined the role of platelet activation in Ang II infusion-induced cardiac inflammation and fibrosis using a P2Y12 receptor inhibitor, clopidogrel (50 mg/[kg·day]). RESULTS: CD41 staining showed that platelets accumulated in Ang II-infused hearts. Clopidogrel treatment inhibited Ang II infusion-induced accumulation of α-SMA(+) myofibroblasts and cardiac fibrosis (4.17 ± 1.26 vs. 1.46 ± 0.81, p < 0.05). Infiltration of inflammatory cells, including Mac-2(+) macrophages and CD45(+)Ly6G(+) neutrophils (30.38 ± 4.12 vs. 18.7 ± 2.38, p < 0.05), into Ang II-infused hearts was also suppressed by platelet inhibition. Real-time PCR and immunohistochemical staining showed that platelet inhibition significantly decreased the expression of interleukin-1ß and transforming growth factor-ß. Acute injection of Ang II or PE stimulated platelet activation and platelet-leukocyte conjugation, which were abolished by clopidogrel treatment. CONCLUSION: Thus, inhibition of platelet activation by clopidogrel prevents cardiac inflammation and fibrosis in response to Ang II. Taken together, our results indicate Ang II infusion-induced hypertension stimulated platelet activation and platelet-leukocyte conjugation, which initiated inflammatory responses that contributed to cardiac fibrosis.

Cathepsin S deficiency results in abnormal accumulation of autophagosomes in macrophages and enhances Ang II-induced cardiac inflammation.

BACKGROUND: Cathepsin S (Cat S) is overexpressed in human atherosclerotic and aneurysmal tissues and may contributes to degradation of extracellular matrix, especially elastin, in inflammatory diseases. We aimed to define the role of Cat S in cardiac inflammation and fibrosis induced by angiotensin II (Ang II) in mice. METHODS AND RESULTS: Cat S-knockout (Cat S(-/-)) and littermate wild-type (WT) C57BL/6J mice were infused continuously with Ang II (750 ng/kg/min) or saline for 7 days. Cat S(-/-) mice showed severe cardiac fibrosis, including elevated expression of collagen I and α-smooth muscle actin (α-SMA), as compared with WT mice. Moreover, macrophage infiltration and expression of inflammatory cytokines (tumor necrosis factor α, transforming growth factor ß and interleukin 1ß) were significantly greater in Cat S(-/-) than WT hearts. These Ang II-induced effects in Cat S(-/-) mouse hearts was associated with abnormal accumulation of autophagosomes and reduced clearance of damaged mitochondria, which led to increased levels of reactive oxygen species (ROS) and activation of nuclear factor-kappa B (NF-κB) in macrophages. CONCLUSION: Cat S in lysosomes is essential for mitophagy processing in macrophages, deficiency in Cat S can increase damaged mitochondria and elevate ROS levels and NF-κB activity in hypertensive mice, so it regulates cardiac inflammation and fibrosis.

Proinflammatory protein CARD9 is essential for infiltration of monocytic fibroblast precursors and cardiac fibrosis caused by Angiotensin II infusion.

BACKGROUND: Angiotensin II (Ang II)-induced cardiac remodeling with the underlying mechanisms involving inflammation and fibrosis has been well documented. Cytosolic adaptor caspase recruitment domain 9 (CARD9) has been implicated in the innate immune response. We aimed to examine the role of CARD9 in inflammation and cardiac fibrosis induced by Ang II. METHODS: Two-month-old CARD9-deficient (CARD9(-/-)) and wild-type (WT) male mice were infused with Ang II (1,500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin and Masson trichrome and examined by immunohistochemistry; and activity and protein levels were measured in macrophages obtained from mice. RESULTS: WT mice with Ang II infusion showed a marked increase in CARD9(+) macrophages in the heart, but CARD9(-/-) mice showed significantly suppressed macrophage infiltration and expression of proinflammatory cytokines, including interleukin-1ß (IL-1ß) and connective tissue growth factor (CTGF). Importantly, Ang II-induced cardiac fibrosis (extracellular matrix and collagen I deposition) was diminished in CARD9(-/-) hearts, as was the expression of transforming growth factor-ß (TGF-ß) and level of myofibroblasts positive for α-smooth muscle actin (α-SMA). Furthermore, Ang II activation of nuclear factor-κB (NF-κB), JNK and p38 mitogen-activated protein kinases (MAPKs) in WT macrophages was reduced in CARD9(-/-) macrophages. CONCLUSION: CARD9 plays an important role in regulating cardiac inflammation and fibrosis in response to elevated Ang II.