Aroylated phenylenediamine HO53 modulates innate immunity, histone acetylation and metabolism.

In the current context of antibiotic resistance, the need to find alternative treatment strategies is urgent. Our research aimed to use synthetized aroylated phenylenediamines (APDs) to induce the expression of cathelicidin antimicrobial peptide gene (CAMP) to minimize the necessity of antibiotic use during infection. One of these compounds, HO53, showed promising results in inducing CAMP expression in bronchial epithelium cells (BCi-NS1.1 hereafter BCi). Thus, to decipher the cellular effects of HO53 on BCi cells, we performed RNA sequencing (RNAseq) analysis after 4, 8 and 24 h treatment of HO53. The number of differentially expressed transcripts pointed out an epigenetic modulation. Yet, the chemical structure and in silico modeling indicated HO53 as a histone deacetylase (HDAC) inhibitor. When exposed to a histone acetyl transferase (HAT) inhibitor, BCi cells showed a decreased expression of CAMP. Inversely, when treated with a specific HDAC3 inhibitor (RGFP996), BCi cells showed an increased expression of CAMP, indicating acetylation status in cells as determinant for the induction of the expression of the gene CAMP expression. Interestingly, a combination treatment with both HO53 and HDAC3 inhibitor RGFP966 leads to a further increase of CAMP expression. Moreover, HDAC3 inhibition by RGFP966 leads to increased expression of STAT3 and HIF1A, both previously demonstrated to be involved in pathways regulating CAMP expression. Importantly, HIF1α is considered as a master regulator in metabolism. A significant number of genes of metabolic enzymes were detected in our RNAseq data with enhanced expression conveying a shift toward enhanced glycolysis. Overall, we are demonstrating that HO53 might have a translational value against infections in the future through a mechanism leading to innate immunity strengthening involving HDAC inhibition and shifting the cells towards an immunometabolism, which further favors innate immunity activation.

Camk2n1 Is a Negative Regulator of Blood Pressure, Left Ventricular Mass, Insulin Sensitivity, and Promotes Adiposity.

Metabolic syndrome is a cause of coronary artery disease and type 2 diabetes mellitus. Camk2n1 resides in genomic loci for blood pressure, left ventricle mass, and type 2 diabetes mellitus, and in the spontaneously hypertensive rat model of metabolic syndrome, Camk2n1 expression is cis-regulated in left ventricle and fat and positively correlates with adiposity. Therefore, we knocked out Camk2n1 in spontaneously hypertensive rat to investigate its role in metabolic syndrome. Compared with spontaneously hypertensive rat, Camk2n1-/- rats had reduced cardiorenal CaMKII (Ca2+/calmodulin-dependent kinase II) activity, lower blood pressure, enhanced nitric oxide bioavailability, and reduced left ventricle mass associated with altered hypertrophic networks. Camk2n1 deficiency reduced insulin resistance, visceral fat, and adipogenic capacity through the altered cell cycle and complement pathways, independent of CaMKII. In human visceral fat, CAMK2N1 expression correlated with adiposity and genomic variants that increase CAMK2N1 expression associated with increased risk of coronary artery disease and type 2 diabetes mellitus. Camk2n1 regulates multiple networks that control metabolic syndrome traits and merits further investigation as a therapeutic target in humans.

Complement Factor B Is a Determinant of Both Metabolic and Cardiovascular Features of Metabolic Syndrome.

CFB (complement factor B) is elevated in adipose tissue and serum from patients with type 2 diabetes mellitus and cardiovascular disease, but the causal relationship to disease pathogenesis is unclear. Cfb is also elevated in adipose tissue and serum of the spontaneously hypertensive rat, a well-characterized model of metabolic syndrome. To establish the role of CFB in metabolic syndrome, we knocked out the Cfb gene in the spontaneously hypertensive rat. Cfb-/- rats showed improved glucose tolerance and insulin sensitivity, redistribution of visceral to subcutaneous fat, increased adipocyte mitochondrial respiration, and marked changes in gene expression. Cfb-/- rats also had lower blood pressure, increased ejection fraction and fractional shortening, and reduced left ventricular mass. These changes in metabolism and gene expression, in adipose tissue and left ventricle, suggest new adipose tissue-intrinsic and blood pressure-independent mechanisms for insulin resistance and cardiac hypertrophy in the spontaneously hypertensive rat. In silico analysis of the human CFB locus revealed 2 cis-regulated expression quantitative trait loci for CFB expression significantly associated with visceral fat, circulating triglycerides and hypertension in genome-wide association studies. Together, these data demonstrate a key role for CFB in the development of spontaneously hypertensive rat metabolic syndrome phenotypes and of related traits in humans and indicate the potential for CFB as a novel target for treatment of cardiometabolic disease.

Genetic, physiological and comparative genomic studies of hypertension and insulin resistance in the spontaneously hypertensive rat.

We previously mapped hypertension-related insulin resistance quantitative trait loci (QTLs) to rat chromosomes 4, 12 and 16 using adipocytes from F2 crosses between spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats, and subsequently identified Cd36 as the gene underlying the chromosome 4 locus. The identity of the chromosome 12 and 16 genes remains unknown. To identify whole-body phenotypes associated with the chromosome 12 and 16 linkage regions, we generated and characterised new congenic strains, with WKY donor segments introgressed onto an SHR genetic background, for the chromosome 12 and 16 linkage regions. We found a >50% increase in insulin sensitivity in both the chromosome 12 and 16 strains. Blood pressure and left ventricular mass were reduced in the two congenic strains consistent with the congenic segments harbouring SHR genes for insulin resistance, hypertension and cardiac hypertrophy. Integrated genomic analysis, using physiological and whole-genome sequence data across 42 rat strains, identified variants within the congenic regions in Upk3bl, RGD1565131 and AABR06087018.1 that were associated with blood pressure, cardiac mass and insulin sensitivity. Quantitative trait transcript analysis across 29 recombinant inbred strains showed correlation between expression of Hspb1, Zkscan5 and Pdgfrl with adipocyte volume, systolic blood pressure and cardiac mass, respectively. Comparative genome analysis showed a marked enrichment of orthologues for human GWAS-associated genes for insulin resistance within the syntenic regions of both the chromosome 12 and 16 congenic intervals. Our study defines whole-body phenotypes associated with the SHR chromosome 12 and 16 insulin-resistance QTLs, identifies candidate genes for these SHR QTLs and finds human orthologues of rat genes in these regions that associate with related human traits. Further study of these genes in the congenic strains will lead to robust identification of the underlying genes and cellular mechanisms.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries.

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries. Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively. SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC. In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation.

Critical role for the advanced glycation end-products receptor in pulmonary arterial hypertension etiology.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This results in both increase in pulmonary arterial pressure and pulmonary vascular resistance. Recent studies have shown the implication of the signal transducer and activator of transcription 3 (STAT3)/bone morphogenetic protein receptor 2 (BMPR2)/peroxisome proliferator-activated receptor gamma (PPARγ) in PAH. STAT3 activation induces BMPR2 downregulation, decreasing PPARγ, which both contribute to the proproliferative and antiapoptotic phenotype seen in PAH. In chondrocytes, activation of this axis has been attributed to the advanced glycation end-products receptor (RAGE). As RAGE is one of the most upregulated proteins in PAH patients' lungs and a strong STAT3 activator, we hypothesized that by activating STAT3, RAGE induces BMPR2 and PPARγ downregulation, promoting PAH-PASMC proliferation and resistance to apoptosis. METHODS AND RESULTS: In vitro, using PASMCs isolated from PAH and healthy patients, we demonstrated that RAGE is overexpressed in PAH-PASMC (6-fold increase), thus inducing STAT3 activation (from 10% to 40% positive cells) and decrease in BMPR2 and PPARγ levels (>50% decrease). Pharmacological activation of RAGE in control cells by S100A4 recapitulates the PAH phenotype (increasing RAGE by 6-fold, thus activating STAT3 and decreasing BMPR2 and PPARγ). In both conditions, this phenotype is totally reversed on RAGE inhibition. In vivo, RAGE inhibition in monocrotaline- and Sugen-induced PAH demonstrates therapeutic effects characterized by PA pressure and right ventricular hypertrophy decrease (control rats have an mPAP around 15 mm Hg, PAH rats have an mPAP >40 mm Hg, and with RAGE inhibition, mPAP decreases to 20 and 28 mm Hg, respectively, in MCT and Sugen models). This was associated with significant improvement in lung perfusion and vascular remodeling due to decrease in proliferation (>50% decrease) and BMPR2/PPARγ axis restoration (increased by ≥60%). CONCLUSION: We have demonstrated the implications of RAGE in PAH etiology. Thus, RAGE constitutes a new attractive therapeutic target for PAH.

Plumbagin reverses proliferation and resistance to apoptosis in experimental PAH.

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

Today's and tomorrow's imaging and circulating biomarkers for pulmonary arterial hypertension.

The pathobiology of pulmonary arterial hypertension (PAH) involves a remodeling process in distal pulmonary arteries, as well as vasoconstriction and in situ thrombosis, leading to an increase in pulmonary vascular resistance, right heart failure and death. Its etiology may be idiopathic, but PAH is also frequently associated with underlying conditions such as connective tissue diseases. During the past decade, more than welcome novel therapies have been developed and are in development, including those increasingly targeting the remodeling process. These therapeutic options modestly increase the patients' long-term survival, now approaching 60% at 5 years. However, non-invasive tools for confirming PAH diagnosis, and assessing disease severity and response to therapy, are tragically lacking and would help to select the best treatment. After exclusion of other causes of pulmonary hypertension, a final diagnosis still relies on right heart catheterization, an invasive technique which cannot be repeated as often as an optimal follow-up might require. Similarly, other techniques and biomarkers used for assessing disease severity and response to treatment generally lack specificity and have significant limitations. In this review, imaging as well as current and future circulating biomarkers for diagnosis, prognosis, and follow-up are discussed.

Krüppel-like factor 5 contributes to pulmonary artery smooth muscle proliferation and resistance to apoptosis in human pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cell (PASMC) and suppressed apoptosis. This phenotype has been associated with the upregulation of the oncoprotein survivin promoting mitochondrial membrane potential hyperpolarization (decreasing apoptosis) and the upregulation of growth factor and cytokines like PDGF, IL-6 and vasoactive agent like endothelin-1 (ET-1) promoting PASMC proliferation. Krüppel-like factor 5 (KLF5), is a zinc-finger-type transcription factor implicated in the regulation of cell differentiation, proliferation, migration and apoptosis. Recent studies have demonstrated the implication of KLF5 in tissue remodeling in cardiovascular diseases, such as atherosclerosis, restenosis, and cardiac hypertrophy. Nonetheless, the implication of KLF5 in pulmonary arterial hypertension (PAH) remains unknown. We hypothesized that KLF5 up-regulation in PAH triggers PASMC proliferation and resistance to apoptosis. METHODS AND RESULTS: We showed that KFL5 is upregulated in both human lung biopsies and cultured human PASMC isolated from distal pulmonary arteries from PAH patients compared to controls. Using stimulation experiments, we demonstrated that PDGF, ET-1 and IL-6 trigger KLF-5 activation in control PASMC to a level similar to the one seen in PAH-PASMC. Inhibition of the STAT3 pathway abrogates KLF5 activation in PAH-PASMC. Once activated, KLF5 promotes cyclin B1 upregulation and promotes PASMC proliferation and triggers survivin expression hyperpolarizing mitochondria membrane potential decreasing PASMC ability to undergo apoptosis. CONCLUSION: We demonstrated for the first time that KLF5 is activated in human PAH and implicated in the pro-proliferative and anti-apoptotic phenotype that characterize PAH-PASMC. We believe that our findings will open new avenues of investigation on the role of KLF5 in PAH and might lead to the identification of new therapeutic targets.

From oncoproteins/tumor suppressors to microRNAs, the newest therapeutic targets for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature characterized by constricted and remodeled pulmonary arteries. This phenomenon is associated with enhanced pulmonary artery smooth muscle cells proliferation and suppressed apoptosis, metabolism shift, inflammation, and several other features that are considered as hallmarks of cancer. Since oncogenes, tumor suppressors, and miRNAs are the major regulators of signaling in the cancer phenotype, we studied if the same type of regulation is operative in PAH. From the discovery of BMPR2 mutation in familial forms of PAH, oncogenic pathways activation like MAPK were identified. Recently, the Src/STAT3/Pim1 axis was also described as playing a critical role in PAH pathogenesis. Moreover, through the down-regulation of miR-204, STAT3 enhances a positive feedback loop sustaining its own activation, showing that miRNA regulation is critical in PAH. Taken together, targeting oncoproteins or miRNAs appear as new therapeutic strategies for PAH. Several oncoprotein inhibitors are already in trials for cancer and could be soon available for PAH. Concerning miRNAs, the youth of this area makes therapies less achievable soon but not less interesting.

RAGE-dependent activation of the oncoprotein Pim1 plays a critical role in systemic vascular remodeling processes.

OBJECTIVE: Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis. Methods/Results- In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator Nε-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model. CONCLUSIONS: RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.