Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway
Mo Ller-Hackbarth, K;Dabaghie, D;Charrin, E;Zambrano, S;Genové, G;Li, X;Wernerson, A;Lal, M;Patrakka, J;
PMID: 34147551 | DOI: 10.1016/j.kint.2021.05.036
Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier, and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.
Hebsgaard JB, Pyke C, Yildirim E, Knudsen LB, Heegaard S, Kvist PH.
PMID: 29707863 | DOI: 10.1111/dom.13339
Semaglutide is a human glucagon-like peptide-1 (GLP-1) analogue that is in development for the treatment of type 2 diabetes. In the pre-approval cardiovascular outcomes trial SUSTAIN 6, semaglutide was associated with a significant increase in the risk of diabetic retinopathy (DR) complications vs placebo. GLP-1 receptor (GLP-1R) expression has previously been demonstrated in the retina in animals and humans; however, antibodies used to detect expression have been documented to be non-specific and fail to detect the GLP-1R using immunohistochemistry (IHC), a problem common for many G-protein coupled receptors. Using a validated GLP-1R antibody for IHC and in situ hybridization for GLP-1R mRNA in normal human eyes, GLP-1Rs were detected in a small fraction of neurons in the ganglion cell layer. In advanced stages of DR, GLP-1R expression was not detected at the protein or mRNA level. Specifically, no GLP-1R expression was found in the eyes of people with long-standing proliferative DR (PDR). In conclusion, GLP-1R expression is low in normal human eyes and was not detected in eyes exhibiting advanced stages of PDR.
Hypertension research : official journal of the Japanese Society of Hypertension
Ochiai, K;Mochida, Y;Nagase, T;Fukuhara, H;Yamaguchi, Y;Nagase, M;
PMID: 36810623 | DOI: 10.1038/s41440-023-01219-9
The recent discovery of mechanosensitive ion channels has promoted mechanobiological research in the field of hypertension and nephrology. We previously reported Piezo2 expression in mouse mesangial and juxtaglomerular renin-producing cells, and its modulation by dehydration. This study aimed to investigate how Piezo2 expression is altered in hypertensive nephropathy. The effects of the nonsteroidal mineralocorticoid receptor blocker, esaxerenone, were also analyzed. Four-week-old Dahl salt-sensitive rats were randomly assigned to three groups: rats fed a 0.3% NaCl diet (DSN), rats fed a high 8% NaCl diet (DSH), and rats fed a high salt diet supplemented with esaxerenone (DSH + E). After six weeks, DSH rats developed hypertension, albuminuria, glomerular and vascular injuries, and perivascular fibrosis. Esaxerenone effectively decreased blood pressure and ameliorated renal damage. In DSN rats, Piezo2 was expressed in Pdgfrb-positive mesangial and Ren1-positive cells. Piezo2 expression in these cells was enhanced in DSH rats. Moreover, Piezo2-positive cells accumulated in the adventitial layer of intrarenal small arteries and arterioles in DSH rats. These cells were positive for Pdgfrb, Col1a1, and Col3a1, but negative for Acta2 (αSMA), indicating that they were perivascular mesenchymal cells different from myofibroblasts. Piezo2 upregulation was reversed by esaxerenone treatment. Furthermore, Piezo2 inhibition by siRNA in the cultured mesangial cells resulted in upregulation of Tgfb1 expression. Cyclic stretch also upregulated Tgfb1 in both transfections of control siRNA and Piezo2 siRNA. Our findings suggest that Piezo2 may have a contributory role in modulating the pathogenesis of hypertensive nephrosclerosis and have also highlighted the therapeutic effects of esaxerenone on salt-induced hypertensive nephropathy. Mechanochannel Piezo2 is known to be expressed in the mouse mesangial cells and juxtaglomerular renin-producing cells, and this was confirmed in normotensive Dahl-S rats. In salt-induced hypertensive Dahl-S rats, Piezo2 upregulation was observed in the mesangial cells, renin cells, and notably, perivascular mesenchymal cells, suggesting its involvement in kidney fibrosis.
Pflugers Archiv : European journal of physiology
Heinl, ES;Broeker, KA;Lehrmann, C;Heydn, R;Krieger, K;Ortmaier, K;Tauber, P;Schweda, F;
PMID: 36480070 | DOI: 10.1007/s00424-022-02774-9
The natriuretic peptides (NPs) ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide) mediate their widespread effects by activating the natriuretic peptide receptor-A (NPR-A), while C-type natriuretic peptide (CNP) acts via natriuretic peptide receptor-B (NPR-B). NPs are removed from the circulation by internalization via the natriuretic peptide clearance receptor natriuretic peptide receptor-C (NPR-C). In addition to their well-known functions, for instance on blood pressure, all three NPs confer significant cardioprotection and renoprotection. Since neither the NP-mediated renal functions nor the renal target cells of renoprotection are completely understood, we performed systematic localization studies of NP receptors using in situ hybridization (RNAscope) in mouse kidneys. NPR-A mRNA is highly expressed in glomeruli (mainly podocytes), renal arterioles, endothelial cells of peritubular capillaries, and PDGFR-receptor β positive (PDGFR-β) interstitial cells. No NPR-A mRNA was detected by RNAscope in the tubular system. In contrast, NPR-B expression is highest in proximal tubules. NPR-C is located in glomeruli (mainly podocytes), in endothelial cells and PDGFR-β positive cells. To test for a possible regulation of NPRs in kidney diseases, their distribution was studied in adenine nephropathy. Signal intensity of NPR-A and NPR-B mRNA was reduced while their spatial distribution was unaltered compared with healthy kidneys. In contrast, NPR-C mRNA signal was markedly enhanced in cell clusters of myofibroblasts in fibrotic areas of adenine kidneys. In conclusion, the primary renal targets of ANP and BNP are glomerular, vascular, and interstitial cells but not the tubular compartment, while the CNP receptor NPR-B is highly expressed in proximal tubules. Further studies are needed to clarify the function and interplay of this specific receptor expression pattern.
Asian Pac J Cancer Prev. 2015;16(11):4549-53.
Raluca BA, Cimpean AM, Cioca A, Cretu O, Mederle O, Ciolofan A, Gaje P, Raica M.
PMID: 26107202
Abstract BACKGROUND: . Colorectal carcinoma (CRC) is one of the major causes of cancer death worldwide. Data from the literature indicate differences between the proliferation rate of endothelial cells relative to the morphology growth type, possibly due to origin of specimens (autopsy material, surgery fragments) or quantification methods. Vascular endothelial growth factor (VEGF) is a factor that stimulates the proliferation of endothelial cells. It is expressed in more than 90% of cases of metastatic CRC. AIM: The aim of this study was to evaluate the endothelial cell proliferation and VEGF expression in primary tumors and corresponding liver metastases. MATERIALS AND METHODS: Our study included 24 recent biopsies of primary tumors and corresponding liver metastases of CRC cases. CD34/ Ki67 double immunostaining and RNA scope assay for VEGF were performed. RESULTS: In the primary tumors analysis of VEGFmRNA expression indicated no significant correlation with differentiation grade, proliferative and non-proliferative vessels in the intratumoral and peritumoral areas. In contrast, in the corresponding liver metastases, VEGFmRNA expression significantly correlated with the total number of non- proliferative vessels and total number of vessels. CD34/ Ki67 double immunostaining in the cases with poorly differentiated carcinoma indicated a high number of proliferating endothelial cells in the peritumoral area and a low number in the intratumoral area for the primary tumor. Moderately differentiated carcinomas of colon showed no proliferating endothelial cells in the intratumoral area in half of the cases included in the study, for both, primary tumor and liver metastasis. In well differentiated CRCs, in primary tumors, a high proliferation rate of endothelial cells in the intratumoral area and a lower proliferation rate in the peritumoral area were found. A low value was found in corresponding liver metastasis. CONCLUSIONS: The absence of proliferative endothelial cells in half of the cases for the primary tumors and liver metastases in moderately differentiated carcinoma suggest a vascular mimicry phenomenon. The mismatch between the total number of vessels and endothelial proliferation in primary tumors indicate that a functional vascular network is already formed or the existence of some mechanisms influenced by other angiogenic factors.
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
Vazquez-Liebanas, E;Nahar, K;Bertuzzi, G;Keller, A;Betsholtz, C;Mäe, MA;
PMID: 34689641 | DOI: 10.1177/0271678X211056395
Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12-18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.
Magno L, Lessard CB, Martins M, Lang V, Cruz P, Asi Y, Katan M, Bilsland J, Lashley T, Chakrabarty P, Golde TE, Whiting PJ.
PMID: 30711010 | DOI: 10.1186/s13195-019-0469-0
Abstract
BACKGROUND:
Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function.
METHODS:
We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays.
RESULTS:
PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function.
CONCLUSIONS:
The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.
Frontiers in molecular biosciences
Nabi, R;Musarrat, F;Menk P Lima, JC;Langohr, IM;Chouljenko, VN;Kousoulas, KG;
PMID: 37388243 | DOI: 10.3389/fmolb.2023.1199068
Introduction: Oncolytic viruses (OVs) provide new modalities for cancer therapy either alone or in combination with synergistic immunotherapies and/or chemotherapeutics. Engineered Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for the treatment of various cancers in experimental animal models as well as in human patients, with some virus strains licensed to treat human melanoma and gliomas. In the present study we evaluated the efficacy of mutant HSV-1 (VC2) in a late stage, highly metastatic 4T1 murine syngeneic. Method: VC2 was constructed VC2 using double red recombination technology. For in-vivo efficacy we utilized a late stage 4T1 syngeneic and immunocompetent BALB/cJ mouse model breast cancer model which exhibits efficient metastasis to the lung and other organs. Results: VC2 replicated efficiently in 4T1 cells and in cell culture, achieving titers similar to those in African monkey kidney (Vero) cells. Intra-tumor treatment with VC2 did not appreciably reduce average primary tumor sizes but a significant reduction of lung metastasis was noted in mice treated intratumorally with VC2, but not with ultraviolet-inactivated VC2. This reduction of metastasis was associated with increased T cell infiltration comprised of CD4+ and CD4+CD8+ double-positive T cells. Characterization of purified tumor infiltrating T cells revealed a significant improvement in their proliferation ability compared to controls. In addition, significant T cell infiltration was observed in the metastatic nodules associated with reduction of pro-tumor PD-L1 and VEGF gene transcription. Conclusion: These results show that VC2 therapy can improve anti-tumor response associated with a better control of tumor metastasis. improve T cell responses and reduce pro-tumor biomarker gene transcription. VC2 holds promise for further development as an oncolytic and immunotherapeutic approach to treat breast and other cancers.
Heydarian, M;Oak, P;Zhang, X;Kamgari, N;Kindt, A;Koschlig, M;Pritzke, T;Gonzalez-Rodriguez, E;Förster, K;Morty, RE;Häfner, F;Hübener, C;Flemmer, AW;Yildirim, AO;Sudheendra, D;Tian, X;Petrera, A;Kirsten, H;Ahnert, P;Morrell, N;Desai, TJ;Sucre, J;Spiekerkoetter, E;Hilgendorff, A;
PMID: 35580897 | DOI: 10.1136/thoraxjnl-2021-218083
Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes.We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice.We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo.We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population.
Distinct Cellular Profiles of Hif1a and Vegf mRNA Localization in Microglia, Astrocytes and Neurons during a Period of Vascular Maturation in the Auditory Brainstem of Neonate Rats
Chang, D;Brown, Q;Tsui, G;He, Y;Liu, J;Shi, L;Rodríguez-Contreras, A;
| DOI: 10.3390/brainsci11070944
Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.
Journal of molecular medicine (Berlin, Germany)
Sych, K;Nold, SP;Pfeilschifter, J;Vutukuri, R;Meisterknecht, J;Wittig, I;Frank, S;Goren, I;
PMID: 36633604 | DOI: 10.1007/s00109-022-02280-6
An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent. KEY MESSAGES: • Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis. • The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia. • In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2. • PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.
Baggio LL, Yusta B, Mulvihill EE, Cao X, Streutker CJ, Butany J, Cappola TP, Margulies KB, Drucker DJ.
PMID: 29444223 | DOI: 10.1210/en.2018-00004
Glucagon-like peptide-1 receptor (GLP-1R) agonists, used to treat type 2 diabetes and obesity, reduce rates of myocardial infarction and cardiovascular death. The GLP-1R has been localized to the human sinoatrial node; however, its expression in ventricular tissue remains uncertain. Here we studied GLP-1R expression in the human heart using GLP-1R-directed antisera, quantitative PCR, reverse transcription PCR to detect full length mRNA transcripts, and in situ hybridization. GLP1R mRNA transcripts, encompassing the entire open reading frame, were detected in all four cardiac chambers from 15 hearts at levels approximating those detected in human pancreas. In contrast, cardiac GLP2R expression was relatively lower, whereas cardiac GCGR expression was sporadic and not detected in the left ventricle. GLP1R mRNA transcripts were not detected in RNA from human cardiac fibroblasts, coronary artery endothelial, or vascular smooth muscle cells. Human Brunner's glands and pancreatic islets exhibited GLP-1R-immunopositivity and abundant expression of GLP1R mRNA transcripts by in situ hybridization. GLP1R transcripts were also detected by in situ hybridization in human cardiac sinoatrial node tissue. However definitive cellular localization of GLP1R mRNA transcripts or immunoreactive GLP-1R protein within human cardiomyocytes (CMs) or cardiac blood vessels remained elusive. Moreover, validated GLP-1R antisera lacked sufficient sensitivity to detect expression of the endogenous islet or cardiac GLP-1R by Western blotting. Hence, although human cardiac ventricles express the GLP1R, the identity of one or more ventricular cell type(s) that express a translated GLP1R protein requires further clarification with highly sensitive methods of detection.
