The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of clusters of differentiation (CD)4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and transforming growth factor β, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using chromatin immunoprecipitation (ChIP), we demonstrate the direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when wild-type Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T-cell component. Significance statement The heat shock response (HSR) is a canonical stress response triggered by a multitude of stressors, including inflammation. Evidence supports the role of the HSR in regulating inflammation, yet there is a paucity of data on its influence in T cells specifically. Gut homeostasis reflects a balance between regulatory clusters of differentiation (CD)4+ T cells and pro-inflammatory T-helper (Th)17 cells. We show that upon activation within T cells, heat shock factor 1 (HSF1) translocates to the nucleus, and stimulates Treg-specific gene expression. HSF1 deficiency hinders Treg development and function and conversely, HSF1 overexpression enhances Treg development and function. While this work, focuses on HSF1 as a novel therapeutic target for intestinal inflammation, the findings have significance for a broad range of inflammatory conditions.

Macrophages are a heterogeneous population with both pro- and anti-inflammatory functions play an essential role in maintaining tissue homeostasis, promoting inflammation under pathological conditions, and tissue repair after injury. In pulmonary hypertension, the M1 phenotype is more pro-inflammatory compared to the M2 phenotype, which is involved in tissue repair. The role of macrophages in the initiation and progression of pulmonary hypertension is well studied. However, their role in the regression of established pulmonary hypertension is not well known. Rats chronically exposed to hemoglobin (Hb) plus hypoxia (HX) share similarities to humans with pulmonary hypertension associated with hemolytic disease, including the presence of a unique macrophage phenotype surrounding distal vessels that are associated with vascular remodeling. These lung macrophages are characterized by high iron content, HO-1, ET-1, and IL-6, and are recruited from the circulation. Depletion of macrophages in this model prevents the development of pulmonary hypertension and vascular remodeling. In this study, we specifically investigate the regression of pulmonary hypertension over a four-week duration after rats were removed from Hb + HX exposure with and without gadolinium chloride administration. Withdrawal of Hb + HX reversed systolic pressures and right ventricular function after Hb + Hx exposure in four weeks. Our data show that depleting circulating monocytes/macrophages during reversal prevents complete recovery of right ventricular systolic pressure and vascular remodeling in this rat model of pulmonary hypertension at four weeks post exposure. The data presented offer a novel insight into the role of macrophages in the processes of pulmonary hypertension regression in a rodent model of Hb + Hx-driven disease.

G protein-coupled receptors (GPCRs) have originally been described as a family of receptors activated by hormones, neurotransmitters, and other mediators. However, in recent years GPCRs have shown to bind endogenous metabolites, which serve functions other than as signaling mediators. These receptors respond to fatty acids, mono- and disaccharides, amino acids, or various intermediates and products of metabolism, including ketone bodies, lactate, succinate, or bile acids. Given that many of these metabolic processes are dysregulated under pathological conditions, including diabetes, dyslipidemia, and obesity, receptors of endogenous metabolites have also been recognized as potential drug targets to prevent and/or treat metabolic and cardiovascular diseases. This review describes G protein-coupled receptors activated by endogenous metabolites and summarizes their physiological, pathophysiological, and potential pharmacological roles.

Pulmonary hypertension (PH) is a progressive cardiovascular disorder in which local vascular inflammation leads to increased pulmonary vascular remodeling and ultimately to right heart failure. The HDAC inhibitor butyrate, a product of microbial fermentation, is protective in inflammatory intestinal diseases, but little is known regarding its effect on extraintestinal diseases, such as PH. In this study, we tested the hypothesis that butyrate is protective in a Sprague–Dawley (SD) rat model of hypoxic PH. Treatment with butyrate (220 mg/kg intake) prevented hypoxia-induced right ventricular hypertrophy (RVH), hypoxia-induced increases in right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, and permeability. A reversal effect of butyrate (2200 mg/kg intake) was observed on elevated RVH. Butyrate treatment also increased the acetylation of histone H3, 25–34 kDa, and 34–50 kDa proteins in the total lung lysates of butyrate-treated animals. In addition, butyrate decreased hypoxia-induced accumulation of alveolar (mostly CD68+) and interstitial (CD68+ and CD163+) lung macrophages. Analysis of cytokine profiles in lung tissue lysates showed a hypoxia-induced upregulation of TIMP-1, CINC-1, and Fractalkine and downregulation of soluble ICAM (sICAM). The expression of Fractalkine and VEGFα, but not CINC-1, TIMP-1, and sICAM was downregulated by butyrate. In rat microvascular endothelial cells (RMVEC), butyrate (1 mM, 2 and 24 h) exhibited a protective effect against TNFα- and LPS-induced barrier disruption. Butyrate (1 mM, 24 h) also upregulated tight junctional proteins (occludin, cingulin, claudin-1) and increased the acetylation of histone H3 but not α-tubulin. These findings provide evidence of the protective effect of butyrate on hypoxic PH and suggest its potential use as a complementary treatment for PH and other cardiovascular diseases.

Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH), a cardiovascular disease. We previously showed that extracellular ATP released under hypoxic conditions is an autocrine/paracrine, the angiogenic factor for pulmonary artery (PA) VV endothelial cells (VVECs), acting via P2Y purinergic receptors (P2YR) and the Phosphoinositide 3-kinase (PI3K)-Akt-Mammalian Target of Rapamycin (mTOR) signaling. To further elucidate the molecular mechanisms of ATP-mediated VV angiogenesis, we determined the profile of ATP-inducible transcription factors (TFs) in VVECs using a TranSignal protein/DNA array. C-Jun, c-Myc, and Foxo3 were found to be upregulated in most VVEC populations and formed nodes connecting several signaling networks. siRNA-mediated knockdown (KD) of these TFs revealed their critical role in ATP-induced VVEC angiogenic responses and the regulation of downstream targets involved in tissue remodeling, cell cycle control, expression of endothelial markers, cell adhesion, and junction proteins. Our results showed that c-Jun was required for the expression of ATP-stimulated angiogenic genes, c-Myc was repressive to anti-angiogenic genes, and Foxo3a predominantly controlled the expression of anti-apoptotic and junctional proteins. The findings from our study suggest that pharmacological targeting of the components of P2YR-PI3K-Akt-mTOR axis and specific TFs reduced ATP-mediated VVEC angiogenic response and may have a potential translational significance in attenuating pathological vascular remodeling.

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a smallinterference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.

senescence, and decreases autophagy in the vasculature [1]. Vascular remodeling and arterial stiffening of large conduit arteries occur due to an increase in collagen deposition and elastin fragmentation resulting in a decrease in compliance. In older arteries, increased activation of RhoA leads to an increase in basal tone in the vasculature. RhoA-ROCK signaling is vital in the expression of contractile proteins in vascular smooth muscle cells. However, sustained RhoGTPase activation leads dedifferentiation of SMC and vascular remodeling [2]. Protein kinase G regulates vascular tone by inhibition of RhoA by phosphorylation. PKG levels decrease with age, which could lead to enhanced vasoconstriction of arteries [1]. Vasoconstriction and impaired vasodilation reduce lumen diameter, increase wall-to-lumen ratio, and inward remodeling of small arteries. Arterial calcification also contributes to vessel stiffness, and is associated with an increase in the transcription factor Runx2, and type I collagen expression in smooth muscle cells. RhoA suppresses the expression of Klotho, a membrane protein associated with longevity [1]. In the Klotho (-/-) aging mouse model, osteoblastlike cells express RUNX-2 in the calcified media, which was attenuated by inhibition of the RhoA-ROCK pathway [3]. Vascular protection due to statins may include their ability to increase Klotho expression. Aging dependent increases in extracellular matrix lead to arterial stiffening in mouse models, which is decreased by inhibitors of RhoA-ROCK signaling. Endothelial dysfunction with age occurs due to increases in vascular permeability, oxidative stress, and inflammation[1]. A decrease in eNOS increases endothelial permeability by increasing Rho activity. Aging increases endothelial cell arginase activity via RhoA, which competes with eNOS for the common substrate L-arginine leading to decreased eNOS-NOvasodilation. Arginase levels are increased in cardiovascular complications of diabetes and hypertension. Studies in animal models indicate that increased ROCK activity causes uncoupling of eNOS, resulting in decreased NO availability and increased reactive nitrogen species and oxidative stress. In diabetic retinopathy, Rac1 activation by Nox2-ROS in the vasculature contributes to mitochondrial damage. In smooth muscle cells, ROS increases levels of cyclophilin Editorial

Inflammation is a well-known feature of heart failure. Studies have shown that while some inflammation is required for repair during injury and is protective, prolonged inflammation leads to myocardial remodeling and apoptosis of cardiac myocytes. Various types of immune cells are implicated in myocardial inflammation and include neutrophils, macrophages, eosinophils, mast cells, natural killer cells, T cells, and B cells. Recent clinical trials have targeted inflammatory cascades as therapy for heart failure with limited success. A better understanding of the temporal course of the infiltration of the different immune cells and their contribution to the inflammatory process may improve the success for therapy. This brief review outlines the major cell types involved in heart failure, and some of their actions are summarized in the supplementary figure.

Ras-homologous (Rho)A/Rho-kinase pathway plays an essential role in many cellular functions, including contraction, motility, proliferation, and apoptosis, inflammation, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Given its role in many physiological and pathological functions, targeting can result in adverse effects and limit its use for therapy. In this review, we have summarized the role of RhoGTPases with an emphasis on RhoA in vascular disease and its impact on endothelial, smooth muscle, and heart and lung fibroblasts. It is clear from the various studies that understanding the regulation of RhoGTPases and their regulators in physiology and pathological conditions is required for effective targeting of Rho.

Pathological vascular remodeling is observed in various cardiovascular diseases including pulmonary hypertension (PH), a disease of unknown etiology that has been characterized by pulmonary artery vasoconstriction, right ventricular hypertrophy, vascular inflammation, and abnormal angiogenesis in pulmonary circulation. G protein-coupled receptors (GPCRs) are the largest family in the genome and widely expressed in cardiovascular system. They regulate all aspects of PH pathophysiology and represent therapeutic targets. We overview GPCRs function in vasoconstriction, vasodilation, vascular inflammation-driven remodeling and describe signaling cross talk between GPCR, inflammatory cytokines, and growth factors. Overall, the goal of this review is to emphasize the importance of GPCRs as critical signal transducers and targets for drug development in PH.

Background and Aims: Inflammatory Bowel Diseases [IBDs] are chronic intestinal inflammatory conditions in part mediated by CD4(+) T cells. Anti-inflammatory Foxp3(+) regulatory T cells [Tregs] maintain immune homeostasis and protect against IBD development via multiple mechanisms, including cytokine secretion and cell-cell interaction. CCAAT enhancer binding protein-beta [C/EBP beta] is a stress-responsive transcription factor linked with IBD susceptibility. Whole-body C/EBP beta deficiency induces CD4(+) T cell-predominant hyperproliferation, and we hypothesize that this may be due to impaired Treg function. Methods: We used the C/EBP beta(-/-) mice in the CD45RB(High) adoptive transfer model, to assess C/EBP beta(-/-) CD4(+) T cells for their colitiogenic potential, and C/EBP beta(-/-) CD4+ Foxp3(+) Tregs for their ability to inhibit colitis. We assessed Tregs from the C/EBP beta(-/-) mice for expression of Treg functional genes and proteins. Results: Naive C/EBP beta(-/-) CD4(+) T cells are more colitogenic in vivo. The exacerbated colitis does not appear to reflect impaired Treg development, however, as C/EBP beta(-/-) mice displayed more, rather than fewer intestinal CD4(+) Foxp3(+) Tregs in vivo. Instead, this reflects impaired Treg function as seen by the reduced capacity to suppress T cell proliferation in vitro, along with decreased secretion of the anti- inflammatory cytokine IL-10. These findings were corroborated in vivo by additional adoptive co- transfer studies in which wildtype Tregs prevented colitis but C/EBP beta(-/-) Tregs did not. Conclusion: C/EBP beta deficiency impairs Treg function and potentiates T cell-mediated colitis. A clearer understanding of the function of this transcription factor may provide a novel therapeutic strategy for IBD.

Angiogenesis is an energy-demanding process; however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose) and oxidative phosphorylation (OXPHOS) (oligomycin, rotenone, and FCCP), we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP, and 2-methylthioadenosine diphosphate trisodium salt (MeSADP), but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase B3, hexokinase (HK), and GLUT-1, but not lactate dehydrogenase. Moreover, extracellular ATP and MeSADP, and to a lesser extent adenosine, increased basal and maximal oxygen consumption rates in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5 mM glucose compared with 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PDH)-E1α and increased succinate dehydrogenase (SDH), cytochrome oxidase IV, and β-subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial Ca2+ concentration, implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of phosphatidylinositol 3-kinase and Akt pathways in lactate production, PDH-E1α phosphorylation, and the expression of HK, SDH, and GLUT-1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways is essential for VV angiogenesis and may contribute to pathologic vascular remodeling in pulmonary hypertension.

Inflammatory bowel diseases are chronic intestinal inflammatory diseases thought to reflect a dysregulated immune response. Although antibody-based inhibition of tumor necrosis factor-α (TNF-α) has provided relief to many inflammatory bowel diseases patients, these therapies are either ineffective in a patient subset or lose their efficacy over time, leaving an unmet need for alternatives. Given the critical role of the heat shock response in regulating inflammation, this study proposed to define the impact of selective inhibition of heat shock protein 90 (HSP90) on intestinal inflammation. Using multiple preclinical mouse models of inflammatory bowel diseases, we demonstrate a potent anti-inflammatory effect of selective inhibition of the HSP90 C-terminal ATPase using the compound novobiocin. Novobiocin-attenuated dextran sulfate sodium-induced colitis and CD45RB adoptive-transfer colitis through the suppression of inflammatory cytokine secretion, including TNF-α. In vitro assays demonstrate that CD4 T cells treated with novobiocin produced significantly less TNF-α measured by intracellular cytokine staining and by enzyme-linked immunosorbent assay. This corresponded to significantly decreased nuclear p65 translocation by Western blot and a decrease in nuclear factor-κB luciferase activity in Jurkat T cells. Finally, to verify the anti-TNF action of novobiocin, 20-week-old TNFΔ mice were treated for 2 weeks with subcutaneous administration of novobiocin. This model has high levels of circulating TNF-α and exhibits spontaneous transmural segmental ileitis. Novobiocin treatment significantly reduced inflammatory cell infiltrate in the ileal lamina propria. HSP90 inhibition with novobiocin offers a novel method of inflammatory cytokine suppression without potential for the development of tolerance that limits current antibody-based methods.

BACKGROUND:The heat shock response is a critical component of the inflammatory cascade designed to prevent misfolding of new proteins and regulate an appropriate immune response. Activation of CD4 T cells causes an up-regulation of the canonical heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that the induction of HSF1 might promote a pro-regulatory phenotype given the appropriate cytokine milieu. METHODS:We aimed to validate this hypothesis using a combination of flow cytometry, immunohistochemistry, western blotting, luciferase assays and chromatin immunoprecipitation (ChIP). In vivo effects were also assessed in adoptive transfer colitis murine models of IBD. RESULTS:Whereas HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and TGFb, a vital cytokine for regulatory T cell (Treg) development, resulted in increased phosphorylation of HSF1 at known activation-associated residues. This in turn led to increased nuclear translocation and binding to heat shock response elements as demonstrated by luciferase assay. Using ChIP, we were able to demonstrate direct binding of HSF1 to the foxp3 promoter in isolated murine CD4 cells, which in turn coincided with induction of Foxp3 expression. Overexpression of HSF1 in transgenic mice led to increased expression of Foxp3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with wildtype CD25+ Tregs at an 8:1 ratio was insufficient to prevent onset of colitis, however, co-transfer of equal numbers of Tregs from mice that over express HSF1 (HSF1-TG), successfully prevented intestinal inflammation. CONCLUSIONS:HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory bowel disease.Funding from CCFA - 253618 (EDZ), K01DK099403-02 (CBC) & CCFA- 253596 (CBC).