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Reelin, a large extracellular glycoprotein, plays critical roles in neuronal development and synaptic plasticity in the central nervous system (CNS). Recent studies have revealed non-neuronal functions of plasma Reelin in inflammation by promoting endothelial-leukocyte adhesion through its canonical pathway in endothelial cells (via ApoER2 acting on NF-κB), as well as in vascular tone regulation and thrombosis. In this study, we have investigated the safety and efficacy of selectively depleting plasma Reelin as a potential therapeutic strategy for chronic inflammatory diseases. We found that Reelin expression remains stable throughout adulthood and that peripheral anti-Reelin antibody treatment with CR-50 efficiently depletes plasma Reelin without affecting its levels or functionality within the CNS. Notably, this approach preserves essential neuronal functions and synaptic plasticity. Furthermore, in mice induced with experimental autoimmune encephalomyelitis (EAE), selective modulation of endothelial responses by anti-Reelin antibodies reduces pathological leukocyte infiltration without completely abolishing diapedesis. Finally, long-term Reelin depletion under metabolic stress induced by a Western diet did not negatively impact the heart, kidney, or liver, suggesting a favorable safety profile. These findings underscore the promising role of peripheral anti-Reelin therapeutic strategies for autoimmune diseases and conditions where endothelial function is compromised, offering a novel approach that may avoid the immunosuppressive side effects associated with conventional anti-inflammatory therapies.

Thromboembolic complications and excessive inflammation are frequent in severe COVID-19, potentially leading to long COVID. In non-COVID studies, we and others demonstrated that circulating Reelin promotes leukocyte infiltration and thrombosis. Thus, we hypothesized that Reelin participates in endothelial dysfunction and hyperinflammation during COVID-19. We showed that Reelin was increased in COVID-19 patients and correlated with the disease activity. In the severe COVID-19 group, we observed a hyperinflammatory state, as judged by increased concentration of cytokines (IL-1α, IL-4, IL-6, IL-10 and IL-17A), chemokines (IP-10 and MIP-1β), and adhesion markers (E-selectin and ICAM-1). Reelin level was correlated with IL-1α, IL-4, IP-10, MIP-1β, and ICAM-1, suggesting a specific role for Reelin in COVID-19 progression. Furthermore, Reelin and all of the inflammatory markers aforementioned returned to normal in a long COVID cohort, showing that the hyperinflammatory state was resolved. Finally, we tested Reelin inhibition with the anti-Reelin antibody CR-50 in hACE2 transgenic mice infected with SARS-CoV-2. CR-50 prophylactic treatment decreased mortality and disease severity in this model. These results demonstrate a direct proinflammatory function for Reelin in COVID-19 and identify it as a drug target. This work opens translational clinical applications in severe SARS-CoV-2 infection and beyond in auto-inflammatory diseases., Competing Interests: LC, MK, and JHE are co-shareholders of Reelin Therapeutics, Inc. LC and JHE are co-inventors of a patent related to anti-Reelin strategies application Number: 15/763,047 and Publication Number: 20180273637. MK was employed by the company Reelin Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Calvier, Drelich, Hsu, Tseng, Mina, Nath, Kounnas and Herz.)

Reelin was originally identified as a regulator of neuronal migration and synaptic function, but its non-neuronal functions have received far less attention. Reelin participates in organ development and physiological functions in various tissues, but it is also dysregulated in some diseases. In the cardiovascular system, Reelin is abundant in the blood, where it contributes to platelet adhesion and coagulation, as well as vascular adhesion and permeability of leukocytes. It is a pro-inflammatory and pro-thrombotic factor with important implications for autoinflammatory and autoimmune diseases such as multiple sclerosis, Alzheimer's disease, arthritis, atherosclerosis, or cancer. Mechanistically, Reelin is a large secreted glycoprotein that binds to several membrane receptors, including ApoER2, VLDLR, integrins, and ephrins. Reelin signaling depends on the cell type but mostly involves phosphorylation of NF-κB, PI3K, AKT, or JAK/STAT. This review focuses on non-neuronal functions and the therapeutic potential of Reelin, while highlighting secretion, signaling, and functional similarities between cell types., Competing Interests: Declaration of interests L.C. and J.H. are co-shareholders of Reelin Therapeutics, Inc. and coinventors of a patent related to anti-Reelin strategies (application 15/763,047 and publication 20180273637)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Under pathological conditions like multiple sclerosis (MS), leukocytes infiltrate the central nervous system where they, in concert with activated microglia, promote inflammatory demyelination resulting in a broad spectrum of symptoms including paralysis. Therefore, all current therapeutic approaches to MS target the immune system, blocking inflammation and paralysis progression, but may compromise the immune system. In this focused review, we present an underestimated compartment, the blood-brain barrier, which is compromised during MS and becomes permeable to leukocytes infiltrating the central nervous system. This barrier has the potential to offer new therapeutic strategies and is easily accessible for drugs. We highlight this paradigm using the example of the therapeutic anti-Reelin strategy we have developed. Reelin is a plasma protein that regulates the expression of adhesion markers on the endothelial surface, thus promoting the infiltration of inflammatory cells and propagating inflammation. Building Back a Better Blood-Brain Barrier (the "6B" strategy) may have advantages compared to actual immunosuppressive drugs because it restores a physiological function rather than suppressing the immune system., Competing Interests: DECLARATION OF INTERESTS L.C. and J.H. are co-shareholders of Reelin Therapeutics Inc. and coinventors of a patent related to anti-Reelin strategies (Application Number: 15/763,047 and Publication Number: 20180273637).

The low-density lipoprotein receptor (LDLR) gene family includes LDLR, very LDLR, and LDL receptor-related proteins (LRPs) such as LRP1, LRP1b (aka LRP-DIT), LRP2 (aka megalin), LRP4, and LRP5/6, and LRP8 (aka ApoER2). LDLR family members constitute a class of closely related multifunctional, transmembrane receptors, with diverse functions, from embryonic development to cancer, lipid metabolism, and cardiovascular homeostasis. While LDLR family members have been studied extensively in the systemic circulation in the context of atherosclerosis, their roles in pulmonary arterial hypertension (PAH) are understudied and largely unknown. Endothelial dysfunction, tissue infiltration of monocytes, and proliferation of pulmonary artery smooth muscle cells are hallmarks of PAH, leading to vascular remodeling, obliteration, increased pulmonary vascular resistance, heart failure, and death. LDLR family members are entangled with the aforementioned detrimental processes by controlling many pathways that are dysregulated in PAH; these include lipid metabolism and oxidation, but also platelet-derived growth factor, transforming growth factor β1, Wnt, apolipoprotein E, bone morpohogenetic proteins, and peroxisome proliferator-activated receptor gamma. In this paper, we discuss the current knowledge on LDLR family members in PAH. We also review mechanisms and drugs discovered in biological contexts and diseases other than PAH that are likely very relevant in the hypertensive pulmonary vasculature and the future care of patients with PAH or other chronic, progressive, debilitating cardiovascular diseases., Competing Interests: Dr Herz was supported by grants from the National Heart, Lung, and Blood Institute (R37 HL063762), the National Institute on Aging (RF AG053391), the National Institute on Neurological Disorders and Stroke and National Institute on Aging (R01 NS093382), and BrightFocus (A2016396S); the Bluefield Project to Cure FTD; and a Harrington Scholar Innovator Award (2019). Dr Hansmann has received financial support from the German Research Foundation (HA4348/2-2 and HA4348/6-2 KFO311), the Federal Ministry of Education and Research (BMBF ViP+ program-03VP08053; BMBF 01KC2001B), and the European Pediatric Pulmonary Vascular Disease Network. Dr Calvier and Dr Herz are shareholders of Reelin Therapeutics and co-inventors of a patent related to anti-Reelin strategies (application number 15/763,047 and publication number 20180273637). Dr Hansmann has reported that he has no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Peroxisome proliferator-activated receptor gamma (PPARγ) is a type II nuclear receptor, initially recognized in adipose tissue for its role in fatty acid storage and glucose metabolism. It promotes lipid uptake and adipogenesis by increasing insulin sensitivity and adiponectin release. Later, PPARγ was implicated in cardiac development and in critical conditions such as pulmonary arterial hypertension (PAH) and kidney failure. Recently, a cluster of different papers linked PPARγ signaling with another superfamily, the transforming growth factor beta (TGFβ), and its receptors, all of which play a major role in PAH and kidney failure. TGFβ is a multifunctional cytokine that drives inflammation, fibrosis, and cell differentiation while PPARγ activation reverses these adverse events in many models. Such opposite biological effects emphasize the delicate balance and complex crosstalk between PPARγ and TGFβ. Based on solid experimental and clinical evidence, the present review summarizes connections and their implications for PAH and kidney failure, highlighting the similarities and differences between lung and kidney mechanisms as well as discussing the therapeutic potential of PPARγ agonist pioglitazone.

The hippocampal formation (HF) is a neuroanatomical region essential for learning and memory. As one of the earliest regions to display the histopathological hallmarks of Alzheimer's disease (AD), determining the specific mechanisms of the HF's vulnerability is of capital importance. Reelin, a glycoprotein crucial in cortical lamination during embryonic neurogenesis, has an uncommon expression pattern within the HF and has been implicated in both learning and AD pathogenesis. We hypothesized that Reelin deficiency would expedite behavioral impairments which accompany normal aging. Additionally, we hypothesized that Reelin deficiency in the presence of mutated human microtubule associated protein tau (MAPT) would further impair hippocampal function. To test our hypothesis, we utilized cohorts of aged mice, aged mice with Reelin conditional knockout (RcKO), and adult mice with both RcKO and MAPT in the Barnes maze and Trace fear conditioning. Consistent with prior literature, increased age in wild-type mice was sufficient to reduce spatial searching in the Barnes maze. Increased age both exacerbated spatial impairments and altered context learning in RcKO mice. Lastly, adult mice with both RcKO and the MAPT transgene displayed both the lowest age-of-onset and most severe spatial learning deficits. In conclusion, Reelin deficiency when combined with AD risk-factors produced consistent impairments in spatial memory tasks. Furthermore, our results further implicate Reelin's importance in both HF homeostasis and AD pathogenesis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Under normal conditions, the blood-brain barrier effectively regulates the passage of immune cells into the central nervous system (CNS). However, under pathological conditions such as multiple sclerosis (MS), leukocytes, especially monocytes, infiltrate the CNS where they promote inflammatory demyelination, resulting in paralysis. Therapies targeting the immune cells directly and preventing leukocyte infiltration exist for MS but may compromise the immune system. Here, we explore how apolipoprotein E receptor 2 (ApoER2) regulates vascular adhesion and infiltration of monocytes during inflammation. We induced experimental autoimmune encephalitis in ApoER2 knockout mice and in mice carrying a loss-of-function mutation in the ApoER2 cytoplasmic domain. In both models, paralysis and neuroinflammation were largely abolished as a result of greatly diminished monocyte adherence due to reduced expression of adhesion molecules on the endothelial surface. Our findings expand our mechanistic understanding of the vascular barrier, the regulation of inflammation and vascular permeability, and the therapeutic potential of ApoER2-targeted therapies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c + cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre +/- ITGA4 fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c + cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c + cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre +/- ITGA4 fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre +/- ITGA4 fl/fl mice, during which CD11c + CD88 + cells were sequestered in the blood. Upon clinical EAE onset, CD11c + CD88 + cells appeared in the CNS and expressed CD317 + In adoptive transfer experiments, CD11c.Cre +/- ITGA4 fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c + CD88 + CD317 + cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice., Competing Interests: Competing interest statement: O.S. serves on the editorial boards of Therapeutic Advances in Neurological Disorders. He has served on data monitoring committees for Genentech-Roche, Pfizer, and TG Therapeutics without monetary compensation. O.S. has advised EMD Serono, Celgene, Genentech, TG Therapeutics, and Genzyme. He currently receives grant support from Sanofi Genzyme and EMD Serono. G.F.W. has received honoraria for consulting for Novartis and Genentech, and received research funding from Biogen, EMD Serono, and Roche. A.H.C. has received honoraria for consulting for Biogen, Celgene, EMD Serono, Genentech, Novartis, Roche, and TG Therapeutics; she has received compensation for speaking for Genentech., (Copyright © 2021 the Author(s). Published by PNAS.)

[Figure: see text].

Neuroinflammation as a result of immune cell recruitment into the central nervous system (CNS) is a key pathogenic mechanism of multiple sclerosis (MS). However, current anti-inflammatory interventions depleting immune cells or directly targeting their trafficking into the CNS can have serious side effects, highlighting a need for better immunomodulatory strategies. We detected increased Reelin concentrations in the serum of patients with MS, resulting in increased endothelial permeability to leukocytes through increased nuclear factor κB-mediated expression of vascular adhesion molecules. We thus investigated the prophylactic and therapeutic potential of Reelin immunodepletion in experimental autoimmune encephalomyelitis (EAE) and further validated the results in Reelin knockout mice. Removal of plasma Reelin by either approach protected against neuroinflammation and largely abolished the neurological consequences by reducing endothelial permeability and immune cell accumulation in the CNS. Our findings suggest Reelin depletion as a therapeutic approach with an inherent good safety margin for the treatment of MS and other diseases where leukocyte extravasation is a major driver of pathogenicity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose of Review: Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial endothelial cell (PAEC) dysfunction and apoptosis, pulmonary arterial smooth muscle cell (PASMC) proliferation, inflammation, vasoconstriction, and metabolic disturbances that include disrupted bone morphogenetic protein receptor (BMPR2)-peroxisome proliferator-activated receptor gamma (PPARγ) axis and DNA damage. Activation of PPARγ improves many of these mechanisms, although erroneous reports on potential adverse effects of thiazolidinedione (TZD)-class PPARγ agonists reduced their clinical use in the past decade. Here, we review recent findings in heart, lung, and kidney research related to the pathobiology of vascular remodeling and tissue fibrosis, and also potential therapeutic effects of the PPARγ agonist pioglitazone., Recent Findings: Independent of its metabolic effects (improved insulin sensitivity and fatty acid handling), PPARγ activation rescues BMPR2 dysfunction, inhibits TGFβ/Smad3/CTGF and TGFβ/pSTAT3/pFoxO1 pathways, and induces the PPARγ/apoE axis, inhibiting vascular remodeling. PPARγ activation dampens mtDNA damage via PPARγ/UBR5/ATM pathway, improves function of endothelial progenitor cells (EPCs), and decrease renal fibrosis by repressing TGFβ/pSTAT3 and TGFβ/EGR1., Summary: Pharmacological PPARγ activation improves many hallmarks of PAH, including dysfunction of BMPR2-PPARγ axis, PAEC, PASMC, EPC, mitochondria/metabolism, and inflammation. Recent randomized controlled trials, including IRIS (Insulin Resistance Intervention After Stroke Trial), emphasize the beneficial effects of PPARγ agonists in PAH patients, leading to recent revival for clinical use.

Translational research is essential to the development of reverse-remodeling strategies for the treatment of pulmonary vascular disease, pulmonary hypertension, and heart failure via mechanistic in vivo studies using animal models resembling human pulmonary arterial hypertension (PAH), cardiovascular remodeling, and progressive right heart failure. Since 2007, peroxisome proliferator-activated receptor γ (PPARγ) agonists have emerged as promising novel, antiproliferative, antiinflammatory, insulin-sensitizing, efficient medications for the treatment of PAH. However, early diabetes study results, their subsequent misinterpretations, errors in published review articles, and rumors regarding potential adverse effects in the literature have dampened enthusiasm for considering pharmacological PPARγ activation for the treatment of cardiovascular diseases, including PAH. Most recently, the thiazolidinedione class PPARγ agonist pioglitazone underwent a clinical revival, especially based on the IRIS (Insulin Resistance Intervention After Stroke) study, a randomized controlled trial in 3,876 patients without diabetes status post-transient ischemic attack/ischemic stroke who were clinically followed for 4.8 years. We discuss preclinical basic translational findings and randomized controlled trials related to the beneficial and adverse effects of PPARγ agonists of the thiazolidinedione class, with a particular focus on the last 5 years. The objective is a data-driven approach to set the preclinical and clinical study record straight. The convincing recent clinical trial data on the lack of significant toxicity in high-risk populations justify the timely conduct of clinical studies to achieve "repurposing" or "repositioning" of pioglitazone for the treatment of clinical PAH.

Background: It has been proposed that peroxisome proliferator-activated receptor-γ (PPARγ) agonists might reduce renal fibrosis, however, several studies had contradictory results. Moreover, the possible interaction of TGF-β 1 , PPARγ, and transcription factors in renal fibrosis have not been investigated. We hypothesized that oral pioglitazone treatment would inhibit TGF-β-driven renal fibrosis and its progression, by modulating profibrotic transcription factors in TGF-β 1 transgenic mice., Methods: Male C57Bl/6 J mice (control, CTL, n = 14) and TGF-β overexpressing transgenic mice (TGFβ, n = 14, having elevated plasma TGF-β 1 level) were divided in two sets at 10 weeks of age. Mice in the first set were fed with regular rodent chow (CTL and TGFβ, n = 7/group). Mice in the second set were fed with chow containing pioglitazone (at a dose of 20 mg/kg/day, CTL + Pio and TGFβ+Pio, n = 7/group). After 5 weeks of treatment, blood pressure was assessed and urine samples were collected, and the kidneys were analyzed for histology, mRNA and protein expression., Results: TGF-β 1 induced glomerulosclerosis and tubulointerstitial damage were significantly reduced by pioglitazone. Pioglitazone inhibited renal mRNA expression of all the profibrotic effectors: type-III collagen, TGF-β 1 , CTGF and TIMP-1, and alike transcription factors cFos/cJun and protein expression of EGR-1, and STAT3 protein phosphorylation., Conclusions: Oral administration of PPARγ agonist pioglitazone significantly reduces TGF-β 1 -driven renal fibrosis, via the attenuation of EGR-1, STAT3 and AP-1. This implies that PPARγ agonists might be effective in the treatment of chronic kidney disease patients.

Rationale: Arterial remodeling-a hallmark of many cardiovascular pathologies including pulmonary arterial hypertension (PAH)-is regulated by TGFβ1 (transforming growth factor-β1)-TGFβ receptors and the antagonistic, vasoprotective BMPR2 (bone morphogenetic protein receptor 2)-PPARγ (peroxisome proliferator-activated receptor-γ) axis. However, it is unclear which factors drive detrimental TGFβ1 pathways in the hypertensive pulmonary vasculature., Objective: We hypothesized that LRP1 (low-density lipoprotein receptor-related protein 1) expression is decreased in PAH, leading to enhancement (disinhibition) of TGFβ1 signals and that the PPARγ agonist pioglitazone can restore vascular homeostasis and prevent PAH resulting from LRP1 deletion in vascular smooth muscle cells (SMCs)., Methods and Results: Targeted deletion of LRP1 in vascular SMC (smLRP1 -/- ) in mice disinhibited TGFβ1-CTGF (connective tissue growth factor) signaling, leading to spontaneous PAH and distal pulmonary arterial muscularization as assessed by closed-chest cardiac catheterization and anti-αSMA staining. Pioglitazone inhibited the canonical TGFβ1-CTGF axis in human pulmonary artery SMC and smLRP1 -/- main pulmonary artery (CTGF and NOX4) and reversed PAH in smLRP1 -/- mice. TGFβ1 boosted pSmad3 in PASMC from smLRP1 -/- mice versus controls. Pioglitazone-activated PPARγ binds to Smad3 in human pulmonary artery SMC (coimmunoprecipitation), thereby blocking its phosphorylation and overriding LRP1 deficiency. Finally, mRNA and protein expression of LRP1 was decreased in pulmonary plexiform lesions of patients with end-stage idiopathic PAH (laser capture microdissection, qPCR, and immunohistochemistry). Downregulation of LRP1 protein was also demonstrated in explanted PASMC from patients with PAH and accompanied by enhanced TGFβ1-pSmad3-CTGF signaling and increased TGFβ1-induced PASMC proliferation that was prevented by pioglitazone., Conclusions: Here, we identify LRP1 as an integrator of TGFβ1-mediated mechanisms that regulate vascular remodeling in mice and clinical PAH and PPARγ as a therapeutic target that controls canonical TGFβ1 pathways. Hence, pharmacologic PPARγ activation represents a promising new therapy for patients with PAH who lack the vasoprotective LRP1 in vascular SMC.

Background: The pharmacological blockade of galectin-3 (Gal-3), a β-galactoside-binding lectin, reduces renal impairment in acute kidney injury, hyperaldosteronism or nephropathy. We herein investigated the effects of pharmacological Gal-3 inhibition by modified citrus pectin (MCP) in renal damage in spontaneously hypertensive rats (SHRs)., Methods and Results: Gal-3 inhibition did not modify blood pressure levels in 30-week-old SHR. Kidney weight was higher in SHR, with no effect of MCP treatment (100 mg/kg/day in the drinking water). Plasma creatinine and albuminuria were slightly but significantly increased in SHR and reduced by MCP, as well as plasma and urinary neutrophil gelatinase-associated lipocalin. In kidney from SHR, Gal-3 was upregulated, as well as the fibrotic markers (collagen type I, TGF-β and connective tissue growth factor) and tubulointerstitial fibrosis. MCP treatment reduced Gal-3 levels and fibrosis. The epithelial-mesenchymal transition (EMT) molecules (fibronectin, α-smooth muscle actin and β-catenin) were modified in SHR and normalized by Gal-3 inhibition. The inflammatory mediators (monocyte chemoattractant protein-1, osteopontin, cd68, cd80, cd44 and cd45) were elevated in SHR and attenuated by MCP. Renal damage markers (neutrophil gelatinase-associated lipocalin and kidney injury molecule-1) were augmented in SHR and improved by MCP. In renal epithelial normal rat kidney-52E cells, Gal-3 treatment induced EMT markers, whereas Gal-3 silencing attenuated EMT., Conclusion: Gal-3 inhibition attenuated early renal damage in SHR as indicated by reduced albuminuria, improved renal function and decreased renal fibrosis, EMT and inflammation, independently of blood pressure levels. These data suggest that Gal-3 could be a potential therapeutic candidate for the prevention of early renal alterations in hypertension.

BMP2 and TGFβ1 are functional antagonists of pathological remodeling in the arteries, heart, and lung; however, the mechanisms in VSMCs, and their disturbance in pulmonary arterial hypertension (PAH), are unclear. We found a pro-proliferative TGFβ1-Stat3-FoxO1 axis in VSMCs, and PPARγ as inhibitory regulator of TGFβ1-Stat3-FoxO1 and TGFβ1-Smad3/4, by physically interacting with Stat3 and Smad3. TGFβ1 induces fibrosis-related genes and miR-130a/301b, suppressing PPARγ. Conversely, PPARγ inhibits TGFβ1-induced mitochondrial activation and VSMC proliferation, and regulates two glucose metabolism-related enzymes, platelet isoform of phosphofructokinase (PFKP, a PPARγ target, via miR-331-5p) and protein phosphatase 1 regulatory subunit 3G (PPP1R3G, a Smad3 target). PPARγ knockdown/deletion in VSMCs activates TGFβ1 signaling. The PPARγ agonist pioglitazone reverses PAH and inhibits the TGFβ1-Stat3-FoxO1 axis in TGFβ1-overexpressing mice. We identified PPARγ as a missing link between BMP2 and TGFβ1 pathways in VSMCs. PPARγ activation can be beneficial in TGFβ1-associated diseases, such as PAH, parenchymal lung diseases, and Marfan's syndrome., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Galectin-3 (Gal-3), a β-galactoside-binding lectin, is increased in kidney injury and its pharmacological blockade reduces renal damage in acute kidney injury, hyperaldosteronism or hypertensive nephropathy. We herein investigated the effects of pharmacological Gal-3 inhibition by modified citrus pectin (MCP) in early renal damage associated with obesity and aortic stenosis (AS)., Results: Gal-3 was upregulated in kidneys from high fat diet (HFD) rats and in animals with partial occlusion of ascending aorta (AS). Urinary and plasma neutrophil gelatinase-associated lipocalin (NGAL) and urinary albumin were enhanced in HFD and AS rats. In kidney from obese rats, fibrotic markers (collagen, TFG-β), epithelial-mesenchymal transition molecules (α-smooth muscle actin, E-cadherin), inflammatory mediator (osteopontin) and kidney injury marker (kidney injury molecule-1) were modified. In kidney from AS rats, fibrotic markers (collagen, CTGF), epithelial-mesenchymal transition molecules (fibronectin, α-smooth muscle actin, β-catenin, E-cadherin) and kidney injury markers (NGAL, kidney injury molecule-1) were altered. Histologic observations of obese and AS rat kidneys revealed tubulointerstitial fibrosis. The pharmacological inhibition of Gal-3 with MCP normalized renal Gal-3 levels as well as functional, histological and molecular alterations in obese and AS rats., Conclusions: In experimental models of mild kidney damage, the increase in renal Gal-3 expression paralleled with renal fibrosis, inflammation and damage, while these alterations were prevented by Gal-3 blockade. These data suggest that Gal-3 could be a new player in renal molecular, histological and functional alterations at early stages of kidney damage., Competing Interests: The authors have declared that no competing interests exist.

Background: Several studies have identified circulating biomarkers to be associated with the presence and severity of pulmonary arterial hypertension (PAH). Recent evidence supports a role for galectin-3 (Gal-3) and the mineralcorticoid aldosterone in left ventricular failure. However, studies on aldosterone together with Gal-3 in PAH are lacking., Objective: We investigated a novel Aldosterone-galectin-3 (Gal-3) tandem and several other potential PAH biomarkers and their association with the disease severity., Methods: A total of 57 patients, 41 with idiopathic PAH. (IPAH) and 16 with PAH associated with connective tissue disease (CTD), and 8 age-matched, non-relative controls were studied. Gal-3, aldosterone and other potential protein plasma concentrations were measured by single ELISA and multi-array MSD (Meso Scale Discovery) technology., Results: Gal-3 values were increased in both patients with IPAH (12.2±0.6 ng/mL; p<0.05) and with PAH-CTD (14.1±1.6 ng/mL; p<0.05) versus control (8.5±0.9 ng/mL), while aldosterone was significantly elevated in IPAH only (248.5±38.8 pg/mL vs control 71.9±18.2 pg/mL; p<0.05). In addition, aldosterone, Gal-3, and N-terminal pro-brain natriuretic peptide (NT-proBNP) values were all higher in patients in WHO functional class II-III versus PAH functional class I or controls. The vascular injury marker intercellular adhesion molecule 1 (ICAM-1) was increased in IPAH and PAH-CTD versus controls (559.5±18.2 pg/mL and 734.1±59.4 pg/mL vs controls 394.8±39.3 pg/mL, p<0.05, p<0.0001, respectively), whereas vascular cell adhesion molecule 1 (VCAM-1) and proinflammatory, anti-angiogenic interleukin-12 (IL-12) were elevated in PAH-CTD only (879.5±110.0 pg/mL and 391.2±70.3 pg/mL vs controls 489.8±44.6 pg/mL, p<0.01, and 102.1±15.2 pg/mL, p<0.01, respectively)., Conclusions: Heightened Gal-3 and aldosterone plasma concentrations in PAH patients indicate a role for Gal-3 signalling in the pathobiology of IPAH and PAH-CTD, and may serve as biomarkers for functional status and progression of disease., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Hypertensive cardiac remodeling is accompanied by molecular inflammation and fibrosis, 2 mechanisms that finally affect cardiac function. At cardiac level, aldosterone promotes inflammation and fibrosis, although the precise mechanisms are still unclear. Galectin-3 (Gal-3), a β-galactoside-binding lectin, is associated with inflammation and fibrosis in the cardiovascular system. We herein investigated whether Gal-3 inhibition could block aldosterone-induced cardiac inflammation and fibrosis and its potential role in cardiac damage associated with hypertension. Aldosterone-salt-treated rats presented hypertension, cardiac inflammation, and fibrosis that were prevented by the pharmacological inhibition of Gal-3 with modified citrus pectin. Cardiac inflammation and fibrosis presented in spontaneously hypertensive rats were prevented by modified citrus pectin treatment, whereas Gal-3 blockade did not modify blood pressure levels. In the absence of blood pressure modifications, Gal-3 knockout mice were resistant to aldosterone-induced cardiac inflammation. In human cardiac fibroblasts, aldosterone increased Gal-3 expression via its mineralocorticoid receptor. Gal-3 and aldosterone enhanced proinflammatory and profibrotic markers, as well as metalloproteinase activities in human cardiac fibroblasts, effects that were not observed in Gal-3-silenced cells treated with aldosterone. In experimental hyperaldosteronism, the increase in Gal-3 expression was associated with cardiac inflammation and fibrosis, alterations that were prevented by Gal-3 blockade independently of blood pressure levels. These data suggest that Gal-3 could be a new molecular mechanism linking cardiac inflammation and fibrosis in situations with high-aldosterone levels, such as hypertension., (© 2015 American Heart Association, Inc.)

Interleukin-33 (IL-33) but not soluble ST2 (sST2) exerts anti-inflammatory and protective effects in several tissues. Aldosterone, a proinflammatory mediator which promotes adipogenesis, is elevated in obese patients. The aim of this study was to investigate the interactions between IL-33/ST2 system and Aldosterone in adipose tissue. Rats fed a high fat diet presented increased sST2 expression, diminished IL-33/sST2 ratio and enhanced levels of differentiation and inflammation in adipose tissue as compared to controls. A similar pattern was observed in adipose tissue from C57BL/6 Aldosterone-treated mice. In both animal models, Aldosterone was correlated with sST2. Treatment of 3T3-L1 adipocytes with IL-33 delayed adipocyte differentiation diminished lipid accumulation and decreased inflammation. Aldosterone decreased IL-33 and increased sST2 expressions in differentiated adipocytes. Aldosterone-induced adipocyte differentiation and inflammation were blocked by IL-33 treatment, but sST2 did not exert any effects. The crosstalk between IL-33/ST2 and Aldosterone could be relevant in the metabolic consequences of obesity., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objectives: This study investigated whether galectin (Gal)-3 inhibition could block aldosterone-induced cardiac and renal fibrosis and improve cardiorenal dysfunction., Background: Aldosterone is involved in cardiac and renal fibrosis that is associated with the development of cardiorenal injury. However, the mechanisms of these interactions remain unclear. Gal-3, a β-galactoside-binding lectin, is increased in heart failure and kidney injury., Methods: Rats were treated with aldosterone-salt combined with spironolactone (a mineralocorticoid receptor antagonist) or modified citrus pectin (a Gal-3 inhibitor), for 3 weeks. Wild-type and Gal-3 knockout mice were treated with aldosterone for 3 weeks. Hemodynamic, cardiac, and renal parameters were analyzed., Results: Hypertensive aldosterone-salt-treated rats presented cardiac and renal hypertrophy (at morphometric, cellular, and molecular levels) and dysfunction. Cardiac and renal expressions of Gal-3 as well as levels of molecular markers attesting fibrosis were also augmented by aldosterone-salt treatment. Spironolactone or modified citrus pectin treatment reversed all of these effects. In wild-type mice, aldosterone did not alter blood pressure levels but increased cardiac and renal Gal-3 expression, fibrosis, and renal epithelial-mesenchymal transition. Gal-3 knockout mice were resistant to aldosterone effects., Conclusions: In experimental hyperaldosteronism, the increase in Gal-3 expression was associated with cardiac and renal fibrosis and dysfunction but was prevented by pharmacological inhibition (modified citrus pectin) or genetic disruption of Gal-3. These data suggest a key role for Gal-3 in cardiorenal remodeling and dysfunction induced by aldosterone. Gal-3 could be used as a new biotarget for specific pharmacological interventions., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: Aldosterone (Aldo) is involved in arterial stiffness and heart failure, but the mechanisms have remained unclear. Galectin-3 (Gal-3), a β-galactoside-binding lectin, plays an important role in inflammation, fibrosis, and heart failure. We investigated here whether Gal-3 is involved in Aldo-induced vascular fibrosis., Methods and Results: In rat vascular smooth muscle cells Gal-3 overexpression enhanced specifically collagen type I synthesis. Moreover Gal-3 inhibition by modified citrus pectin or small interfering RNA blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, inflammation, fibrosis, and increased aortic Gal-3 expression. Spironolactone or modified citrus pectin treatment reversed all the above effects. Wild-type and Gal-3 knock-out mice were treated with Aldo for 6 hours or 3 weeks. Aldo increased aortic Gal-3 expression, inflammation, and collagen type I in wild-type mice at both the short- and the long-term, whereas no changes occurred in Gal-3 knock-out mice., Conclusions: Our data indicate that Gal-3 is required for inflammatory and fibrotic responses to Aldo in vascular smooth muscle cells in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis.

Cardiotrophin 1 (CT-1), an interleukin 6 family member, promotes fibrosis and arterial stiffness. We hypothesized that the absence of CT-1 influences arterial fibrosis and stiffness, senescence, and life span. In senescent 29-month-old mice, vascular function was analyzed by echotracking device. Arterial histomorphology, senescence, metabolic, inflammatory, and oxidative stress parameters were measured by immunohistochemistry, reverse transcription polymerase chain reaction, Western blot, and ELISA. Survival rate of wild-type and CT-1-null mice was studied. Vascular smooth muscle cells were treated with CT-1 (10(-9) mol/L) for 15 days to analyze senescence. The wall stress-incremental elastic modulus curve of old CT-1-null mice was shifted rightward as compared with wild-type mice, indicating decreased arterial stiffness. Media thickness and wall fibrosis were lower in CT-1-null mice. CT-1-null mice showed decreased levels of inflammatory, apoptotic, and senescence pathways, whereas telomere-linked proteins, DNA repair proteins, and antioxidant enzyme activities were increased. CT-1-null mice displayed a 5-month increased median longevity compared with wild-type mice. In vascular smooth muscle cells, chronic CT-1 stimulation upregulated apoptotic and senescence markers and downregulated telomere-linked proteins. The absence of CT-1 is associated with decreased arterial fibrosis, stiffness, and senescence and increased longevity in mice likely through downregulating apoptotic, senescence, and inflammatory pathways. CT-1 may be a major regulator of arterial stiffness with a major impact on the aging process.

Cardiotrophin 1 (CT-1), a cytokine belonging to the interleukin 6 family, is increased in hypertension and in heart failure. We aimed to study the precise role of CT-1 on cardiac, vascular, and renal function; morphology; and remodeling in early stages without hypertension. CT-1 (20 μg/kg per day) or vehicle was administrated to Wistar rats for 6 weeks. Cardiac and vascular functions were analyzed in vivo using M-mode echocardiography, Doppler, and echo tracking device and ex vivo using a scanning acoustic microscopy method. Cardiovascular and renal histomorphology were measured by immunohistochemistry, RT-PCR, and Western blot. Kidney functional properties were assessed by serum creatinine and neutrophile gelatinase-associated lipocalin and microalbuminuria/creatininuria ratio. Without alterations in blood pressure levels, CT-1 treatment increased left ventricular volumes, reduced fractional shortening and ejection fraction, and induced myocardial dilatation and myocardial fibrosis. In the carotid artery of CT-1-treated rats, the circumferential wall stress-incremental elastic modulus curve was shifted leftward, and the acoustic speed of sound in the aorta was augmented, indicating increased arterial stiffness. Vascular media thickness, collagen, and fibronectin content were increased by CT-1 treatment. CT-1-treated rats presented unaltered serum creatinine concentrations but increased urinary and serum neutrophile gelatinase-associated lipocalin and microalbuminuria/creatininuria ratio. This paralleled a glomerular and tubulointerstitial fibrosis accompanied by renal epithelial-mesenchymal transition. CT-1 is a new potent fibrotic agent in heart, vessels, and kidney able to induce cardiovascular-renal dysfunction independent from blood pressure. Thus, CT-1 could be a new target simultaneously integrating alterations of heart, vessels, and kidney in early stages of heart failure.