Leukocyte Overexpression of Intracellular NAMPT Attenuates Atherosclerosis by Regulating PPARγ-Dependent Monocyte Differentiation and Function

Objective-Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) mediates inflammatory and potentially proatherogenic effects, whereas the role of intracellular NAMPT (iNAMPT), the rate limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD)+ generation, in atherogenesis is largely unknown. Here we investigated the effects of iNAMPT overexpression in leukocytes on inflammation and atherosclerosis. Approach and Results-Low-density lipoprotein receptor-deficient mice with hematopoietic overexpression of human iNAMPT (iNAMPThi), on a western type diet, showed attenuated plaque burden with features of lesion stabilization. This anti-atherogenic effect was caused by improved resistance of macrophages to apoptosis by attenuated chemokine (C-C motif) receptor 2-dependent monocyte chemotaxis and by skewing macrophage polarization toward an anti-inflammatory M2 phenotype. The iNAMPThi phenotype was almost fully reversed by treatment with the NAMPT inhibitor FK866, indicating that iNAMPT catalytic activity is instrumental in the atheroprotection. Importantly, iNAMPT overexpression did not induce any increase in eNAMPT, and eNAMPT had no effect on chemokine (C-C motif) receptor 2 expression and promoted an inflammatory M1 phenotype in macrophages. The iNAMPT-mediated effects at least partly involved sirtuin 1-dependent molecular crosstalk of NAMPT and peroxisome proliferator-activated receptor γ. Finally, iNAMPT and peroxisome proliferator-activated receptor γ showed a strong correlation in human atherosclerotic, but not healthy arteries, hinting to a relevance of iNAMPT/peroxisome proliferator-activated receptor γ pathway also in human carotid atherosclerosis. Conclusions-This study highlights the functional dichotomy of intracellular versus extracellular NAMPT, and unveils a critical role for the iNAMPT-peroxisome proliferator-activated receptor γ axis in atherosclerosis.Netherlands Heart Foundation 2003T201European Union FP7–PEOPLE–2007–2–1–IEF, FP7– PEOPLE–2010–RGMinisterio de Economía y Competitividad AGL2011–2900

Leukocyte Overexpression of Intracellular NAMPT Attenuates Atherosclerosis by Regulating PPAR gamma-Dependent Monocyte Differentiation and Function

Objective-Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) mediates inflammatory and potentially proatherogenic effects, whereas the role of intracellular NAMPT (iNAMPT), the rate limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD)(+) generation, in atherogenesis is largely unknown. Here we investigated the effects of iNAMPT overexpression in leukocytes on inflammation and atherosclerosis. Approach and Results-Low-density lipoprotein receptor-deficient mice with hematopoietic overexpression of human iNAMPT (iNAMPT(hi)), on a western type diet, showed attenuated plaque burden with features of lesion stabilization. This anti-atherogenic effect was caused by improved resistance of macrophages to apoptosis by attenuated chemokine (C-C motif) receptor 2-dependent monocyte chemotaxis and by skewing macrophage polarization toward an anti-inflammatory M2 phenotype. The iNAMPT(hi) phenotype was almost fully reversed by treatment with the NAMPT inhibitor FK866, indicating that iNAMPT catalytic activity is instrumental in the atheroprotection. Importantly, iNAMPT overexpression did not induce any increase in eNAMPT, and eNAMPT had no effect on chemokine (C-C motif) receptor 2 expression and promoted an inflammatory M1 phenotype in macrophages. The iNAMPT-mediated effects at least partly involved sirtuin 1-dependent molecular crosstalk of NAMPT and peroxisome proliferator-activated receptor.. Finally, iNAMPT and peroxisome proliferator-activated receptor. showed a strong correlation in human atherosclerotic, but not healthy arteries, hinting to a relevance of iNAMPT/peroxisome proliferator-activated receptor. pathway also in human carotid atherosclerosis. Conclusions-This study highlights the functional dichotomy of intracellular versus extracellular NAMPT, and unveils a critical role for the iNAMPT-peroxisome proliferator-activated receptor. axis in atherosclerosis

Macrophage-specific expression of mannose-binding lectin controls atherosclerosis in low-density lipoprotein receptor-deficient mice

With consideration of the central role of the innate immune system in atherogenesis and mannose-binding lectin (MBL) as an innate regulator of immunity, the role of MBL in experimental and human atherosclerosis was assessed. With the use of immunohistochemistry and polymerase chain reaction, deposition and gene expression of MBL-A and -C were assessed in murine atherosclerosis from mice deficient for the low-density lipoprotein receptor (LDLR(-/-)) after 10 or 18 weeks of high-fat feeding. MBL was present and was produced in 10-week-old lesions, whereas deposition and gene expression were minimal after 18 weeks of high-fat feeding and absent in healthy vasculature. Interestingly, deposition of MBL-A and -C differed: MBL-A predominantly localized in upper medial layers, whereas MBL-C was found in and around intimal macrophages. To further study the role of local MBL production by monocytic cells in atherosclerosis, LDLR(-/-) mice with MBL-A and -C(-/-) monocytic cells were construed by bone marrow transplantation. Mice carrying MBL-A and -C double deficient macrophages had increased (30%) atherosclerotic lesions compared with wild-type controls (P=0.015) after 10 weeks of high-fat diet. Subsequently, analysis of MBL deposition and gene expression in advanced human atherosclerotic lesions revealed the presence of MBL protein in ruptured but not stable atherosclerotic lesions. Putatively in agreement with murine data, no MBL gene expression could be detected in advanced human atherosclerotic lesions. These results are the first to show that MBL is abundantly present and locally produced during early atherogenesis. Local MBL expression, by myeloid cells, is shown to critically control development of atherosclerotic lesion

Cathepsin K gene disruption does not affect murine aneurysm formation

Cathepsin K (catK), a lysosomal cysteine protease, exerts strong elastinolytic and collagenolytic activity and is implicated in a range of pathological disorders including cardiovascular disease. CatK expression was found to be elevated in human aortic aneurysm pointing to a role in this vasculopathy. In the angiotensin II (Ang II)-induced mouse model for aneurysm formation, catK, S and C expression was strongly upregulated. Therefore, we investigated the effect of catK deficiency on Ang II-induced aneurysm formation in the abdominal aorta of apoE-/- mice. Contrary to our expectations, catK deficiency did not protect against aneurysm formation, nor did it affect medial elastin breaks. Proteolytic activity in abdominal aortic lysates were comparable between apoE-/- and catK-//-apoE-/- mice. Adventitial presence of catS- and catC-expressing cells was significantly increased in catK-/-//apoE-/- versus apoE-/- mice, which might have compensated for the deficiency of catK-derived proteolysis in the aneurysm tissue of catK deficient apoE-/- mice. Circulating granulocytes and activated T cell numbers were significantly increased in Ang II-infused catK-/-//apoE-/- mice, which is consistent with the borderline significant increase in adventitial leukocyte content in catK-/-//apoE-/- compared to apoE-/- mice. Strikingly, despite unchanged proteolytic activity in AAA lesions, collagen content in the aneurysm was significantly increased in catK-//-apoE-/- mice. In conclusion, while catK deficiency has major impact on various vasculopathies, it did not affect murine aneurysm formatio

Increased Expression of Syndecan-1 Protects Against Cardiac Dilatation and Dysfunction After Myocardial Infarction

BACKGROUND: The cell-associated proteoglycan syndecan-1 (Synd1) closely regulates inflammation and cell-matrix interactions during wound healing and tumorigenesis. The present study investigated whether Synd1 may also regulate cardiac inflammation, matrix remodeling, and function after myocardial infarction (MI). METHODS AND RESULTS: First, we showed increased protein and mRNA expression of Synd1 from 24 hours on, reaching its maximum at 7 days after MI and declining thereafter. Targeted deletion of Synd1 resulted in increased inflammation and accelerated, yet functionally adverse, infarct healing after MI. In concordance, adenoviral gene expression of Synd1 protected against exaggerated inflammation after MI, mainly by reducing transendothelial adhesion and migration of leukocytes, as shown in vitro. Increased inflammation in the absence of Synd1 resulted in increased monocyte chemoattractant protein-1 expression, increased activity of matrix metalloproteinase-2 and -9, and decreased activity of tissue transglutaminase, associated with increased collagen fragmentation and disorganization. Exaggerated inflammation and adverse matrix remodeling in the absence of Synd1 increased cardiac dilatation and impaired systolic function, whereas gene overexpression of Synd1 reduced inflammation and protected against cardiac dilatation and failure. CONCLUSIONS: Increased expression of Synd1 in the infarct protects against exaggerated inflammation and adverse infarct healing, thereby reducing cardiac dilatation and dysfunction after MI in mice.status: publishe

Increased expression of syndecan-1 protects against cardiac dilatation and dysfunction after myocardial infarction

BACKGROUND: The cell-associated proteoglycan syndecan-1 (Synd1) closely regulates inflammation and cell-matrix interactions during wound healing and tumorigenesis. The present study investigated whether Synd1 may also regulate cardiac inflammation, matrix remodeling, and function after myocardial infarction (MI). METHODS AND RESULTS: First, we showed increased protein and mRNA expression of Synd1 from 24 hours on, reaching its maximum at 7 days after MI and declining thereafter. Targeted deletion of Synd1 resulted in increased inflammation and accelerated, yet functionally adverse, infarct healing after MI. In concordance, adenoviral gene expression of Synd1 protected against exaggerated inflammation after MI, mainly by reducing transendothelial adhesion and migration of leukocytes, as shown in vitro. Increased inflammation in the absence of Synd1 resulted in increased monocyte chemoattractant protein-1 expression, increased activity of matrix metalloproteinase-2 and -9, and decreased activity of tissue transglutaminase, associated with increased collagen fragmentation and disorganization. Exaggerated inflammation and adverse matrix remodeling in the absence of Synd1 increased cardiac dilatation and impaired systolic function, whereas gene overexpression of Synd1 reduced inflammation and protected against cardiac dilatation and failure. CONCLUSIONS: Increased expression of Synd1 in the infarct protects against exaggerated inflammation and adverse infarct healing, thereby reducing cardiac dilatation and dysfunction after MI in mic

Regulation of Cardiac Gene Expression by KLF15, a Repressor of Myocardin Activity*

Pathological forms of left ventricular hypertrophy (LVH) often progress to heart failure. Specific transcription factors have been identified that activate the gene program to induce pathological forms of LVH. It is likely that apart from activating transcriptional inducers of LVH, constitutive transcriptional repressors need to be removed during the development of cardiac hypertrophy. Here, we report that the constitutive presence of Krüppel-like factor 15 (KLF15) is lost in pathological hypertrophy and that this loss precedes progression toward heart failure. We show that transforming growth factor-β-mediated activation of p38 MAPK is necessary and sufficient to decrease KLF15 expression. We further show that KLF15 robustly inhibits myocardin, a potent transcriptional activator. Loss of KLF15 during pathological LVH relieves the inhibitory effects on myocardin and stimulates the expression of serum response factor target genes, such as atrial natriuretic factor. This uncovers a novel mechanism where activated p38 MAPK decreases KLF15, an important constitutive transcriptional repressor whose removal seems a vital step to allow the induction of pathological LVH

miR-133 and miR-30 Regulate Connective Tissue Growth Factor Implications for a Role of MicroRNAs in Myocardial Matrix Remodeling

The myocardium of the failing heart undergoes a number of structural alterations, most notably hypertrophy of cardiac myocytes and an increase in extracellular matrix proteins, often seen as primary fibrosis. Connective tissue growth factor (CTGF) is a key molecule in the process of fibrosis and therefore seems an attractive therapeutic target. Regulation of CTGF expression at the promoter level has been studied extensively, but it is unknown how CTGF transcripts are regulated at the posttranscriptional level. Here we provide several lines of evidence to show that CTGF is importantly regulated by 2 major cardiac microRNAs (miRNAs), miR-133 and miR-30. First, the expression of both miRNAs was inversely related to the amount of CTGF in 2 rodent models of heart disease and in human pathological left ventricular hypertrophy. Second, in cultured cardiomyocytes and fibroblasts, knockdown of these miRNAs increased CTGF levels. Third, overexpression of miR-133 or miR-30c decreased CTGF levels, which was accompanied by decreased production of collagens. Fourth, we show that CTGF is a direct target of these miRNAs, because they directly interact with the 3' untranslated region of CTGF. Taken together, our results indicate that miR-133 and miR-30 importantly limit the production of CTGF. We also provide evidence that the decrease of these 2 miRNAs in pathological left ventricular hypertrophy allows CTGF levels to increase, which contributes to collagen synthesis. In conclusion, our results show that both miR-133 and miR-30 directly downregulate CTGF, a key profibrotic protein, and thereby establish an important role for these miRNAs in the control of structural changes in the extracellular matrix of the myocardium. (Circ Res. 2009; 104: 170-178.