Predicted Value of MicroRNAs, Vascular Endothelial Growth Factor, and Intermediate Monocytes in the Left Adverse Ventricular Remodeling in Revascularized ST-Segment Elevation Myocardial Infarction Patients.

Background: Primary percutaneous coronary intervention (PPCI) in patients with ST-segment elevation myocardial infarction (STEMI) improves the survival of patients; nevertheless, some patients develop left ventricular adverse remodeling (LVAR) a few months after the intervention. The main objective of this study was to characterize the role of pro-inflammatory cell populations, related cytokines, and microRNAs (miRNAs) released after PPCI as reliable prognostic biomarkers for LVAR in patients with STEMI. Methods: We evaluated the level of pro-inflammatory subsets, before and after revascularization, 1 and 6 months after PPCI, using flow cytometry. We also performed a miRNA microarray in isolated peripheral blood mononuclear cells (PBMCs) and examined the levels of 27 cytokines in patients' serum of patients by multiplex ELISA. Results: We observed that the levels of classical and intermediate monocytes increased 6 h after PPCI in patients who developed LVAR later. Multivariate regression analysis and ROC curves indicated that intermediate monocytes, after PPCI, were the best monocyte subset that correlated with LVAR. Within the 27 evaluated cytokines evaluated, we found that the increase in the level of vascular endothelial growth factor (VEGF) correlated with LVAR. Furthermore, the microarray analysis of PBMCs determined that up to 1,209 miRNAs were differentially expressed 6 h after PPCI in LVAR patients, compared with those who did not develop LVAR. Using RT-qPCR we confirmed a significant increase in miR-16, miR-21-5p, and miR-29a-3p, suggested to modulate the expression of different cytokines, 6 h post-PPCI in LVAR patients. Interestingly, we determined that the combined analysis of the levels of the intermediate monocyte subpopulation, VEGF, and miRNAs gave a better association with LVAR appearance. Similarly, combined ROC analysis provided high accurate specificity and sensibility to identify STEMI patients who will develop LVAR. Conclusion: Our data suggest that the combined analysis of intermediate monocytes, VEGF, and miRNAs predicts LVAR in STEMI patients.

Heterotopic ossification in mice overexpressing Bmp2 in Tie2+ lineages.

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation.

Potential Tamoxifen Repurposing to Combat Infections by Multidrug-Resistant Gram-Negative Bacilli.

The development of new strategic therapies for multidrug-resistant bacteria, like the use of non-antimicrobial approaches and/or drugs repurposed to be used as monotherapies or in combination with clinically relevant antibiotics, has become urgent. A therapeutic alternative for infections by multidrug-resistant Gram-negative bacilli (MDR-GNB) is immune system modulation to improve the infection clearance. We showed that immunocompetent mice pretreated with tamoxifen at 80 mg/kg/d for three days and infected with Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli in peritoneal sepsis models showed reduced release of the monocyte chemotactic protein-1 (MCP-1) and its signaling pathway interleukin-18 (IL-18), and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). This reduction of MCP-1 induced the reduction of migration of inflammatory monocytes and neutrophils from the bone marrow to the blood. Indeed, pretreatment with tamoxifen in murine peritoneal sepsis models reduced the bacterial load in tissues and blood, and increased mice survival from 0% to 60-100%. Together, these data show that tamoxifen presents therapeutic efficacy against MDR A. baumannii, P. aeruginosa, and E. coli in experimental models of infection and may be a new candidate to be repurposed as a treatment for GNB infections.

Corrigendum: SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.

[This corrects the article DOI: 10.3389/fcell.2021.639952.].

Non-productive angiogenesis disassembles Aß plaque-associated blood vessels.

The human Alzheimer's disease (AD) brain accumulates angiogenic markers but paradoxically, the cerebral microvasculature is reduced around Aß plaques. Here we demonstrate that angiogenesis is started near Aß plaques in both AD mouse models and human AD samples. However, endothelial cells express the molecular signature of non-productive angiogenesis (NPA) and accumulate, around Aß plaques, a tip cell marker and IB4 reactive vascular anomalies with reduced NOTCH activity. Notably, NPA induction by endothelial loss of presenilin, whose mutations cause familial AD and which activity has been shown to decrease with age, produced a similar vascular phenotype in the absence of Aß pathology. We also show that Aß plaque-associated NPA locally disassembles blood vessels, leaving behind vascular scars, and that microglial phagocytosis contributes to the local loss of endothelial cells. These results define the role of NPA and microglia in local blood vessel disassembly and highlight the vascular component of presenilin loss of function in AD.

SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.

Angiogenesis is a multistep process that controls endothelial cells (ECs) functioning to form new blood vessels from preexisting vascular beds. This process is tightly regulated by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which promote signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recent evidence suggests that store-operated calcium entry (SOCE) might play a role in angiogenesis. However, little is known regarding the role of SARAF, SOCE-associated regulatory factor, and Orai1, the pore-forming subunit of the store-operated calcium channel (SOCC), in angiogenesis. Here, we show that SOCE inhibition with GSK-7975A blocks aorta sprouting, as well as human umbilical vein endothelial cell (HUVEC) tube formation and migration. The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice, since it reduces vessel length and the number of junctions, while it increases lacunarity. Moreover, we find that SARAF and Orai1 are involved in VEGF-mediated [Ca2+]i increase, and their knockdown using siRNA impairs HUVEC tube formation, proliferation, and migration. Finally, immunostaining and in situ proximity ligation assays indicate that SARAF likely interacts with Orai1 in HUVECs. Therefore, these findings show for the first time a functional interaction between SARAF and Orai1 in ECs and highlight their essential role in different steps of the angiogenesis process.

TRPC and TRPV Channels' Role in Vascular Remodeling and Disease.

Transient receptor potentials (TRPs) are non-selective cation channels that are widely expressed in vascular beds. They contribute to the Ca2+ influx evoked by a wide spectrum of chemical and physical stimuli, both in endothelial and vascular smooth muscle cells. Within the superfamily of TRP channels, different isoforms of TRPC (canonical) and TRPV (vanilloid) have emerged as important regulators of vascular tone and blood flow pressure. Additionally, several lines of evidence derived from animal models, and even from human subjects, highlighted the role of TRPC and TRPV in vascular remodeling and disease. Dysregulation in the function and/or expression of TRPC and TRPV isoforms likely regulates vascular smooth muscle cells switching from a contractile to a synthetic phenotype. This process contributes to the development and progression of vascular disorders, such as systemic and pulmonary arterial hypertension, atherosclerosis and restenosis. In this review, we provide an overview of the current knowledge on the implication of TRPC and TRPV in the physiological and pathological processes of some frequent vascular diseases.

Circulating miR-320a as a Predictive Biomarker for Left Ventricular Remodelling in STEMI Patients Undergoing Primary Percutaneous Coronary Intervention.

Restoration of epicardial coronary blood flow, achieved by early reperfusion with primary percutaneous coronary intervention (PPCI), is the guideline recommended to treat patients with ST-segment-elevation myocardial infarction (STEMI). However, despite successful blood restoration, increasing numbers of patients develop left ventricular adverse remodelling (LVAR) and heart failure. Therefore, reliable prognostic biomarkers for LVAR in STEMI are urgently needed. Our aim was to investigate the role of circulating microRNAs (miRNAs) and their association with LVAR in STEMI patients following the PPCI procedure. We analysed the expression of circulating miRNAs in blood samples of 56 patients collected at admission and after revascularization (at 3, 6, 12 and 24 h). The associations between miRNAs and left ventricular end diastolic volumes at 6 months were estimated to detect LVAR. miRNAs were also analysed in samples isolated from peripheral blood mononuclear cells (PBMCs) and human myocardium of failing hearts. Kinetic analysis of miRNAs showed a fast time-dependent increase in miR-133a, miR-133b, miR-193b, miR-499, and miR-320a in STEMI patients compared to controls. Moreover, the expression of miR-29a, miR-29b, miR-324, miR-208, miR-423, miR-522, and miR-545 was differentially expressed even before PPCI in STEMI. Furthermore, the increase in circulating miR-320a and the decrease in its expression in PBMCs were significantly associated with LVAR and correlated with the expression of miR-320a in human failing myocardium from ischaemic origin. In conclusion, we determined the time course expression of new circulating miRNAs in patients with STEMI treated with PPCI and we showed that miR-320a was positively associated with LVAR.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.

TRP Channels: Current Perspectives in the Adverse Cardiac Remodeling.

Calcium is an important second messenger required not only for the excitation-contraction coupling of the heart but also critical for the activation of cell signaling pathways involved in the adverse cardiac remodeling and consequently for the heart failure. Sustained neurohumoral activation, pressure-overload, or myocardial injury can cause pathologic hypertrophic growth of the heart followed by interstitial fibrosis. The consequent heart's structural and molecular adaptation might elevate the risk of developing heart failure and malignant arrhythmia. Compelling evidences have demonstrated that Ca2+ entry through TRP channels might play pivotal roles in cardiac function and pathology. TRP proteins are classified into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), and TRPP (polycystin), which are activated by numerous physical and/or chemical stimuli. TRP channels participate to the handling of the intracellular Ca2+ concentration in cardiac myocytes and are mediators of different cardiovascular alterations. This review provides an overview of the current knowledge of TRP proteins implication in the pathologic process of some frequent cardiac diseases associated with the adverse cardiac remodeling such as cardiac hypertrophy, fibrosis, and conduction alteration.

Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate.

The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

Nestin(+) cells direct inflammatory cell migration in atherosclerosis.

Atherosclerosis is a leading death cause. Endothelial and smooth muscle cells participate in atherogenesis, but it is unclear whether other mesenchymal cells contribute to this process. Bone marrow (BM) nestin(+) cells cooperate with endothelial cells in directing monocyte egress to bloodstream in response to infections. However, it remains unknown whether nestin(+) cells regulate inflammatory cells in chronic inflammatory diseases, such as atherosclerosis. Here, we show that nestin(+) cells direct inflammatory cell migration during chronic inflammation. In Apolipoprotein E (ApoE) knockout mice fed with high-fat diet, BM nestin(+) cells regulate the egress of inflammatory monocytes and neutrophils. In the aorta, nestin(+) stromal cells increase ∼30 times and contribute to the atheroma plaque. Mcp1 deletion in nestin(+) cells-but not in endothelial cells only- increases circulating inflammatory cells, but decreases their aortic infiltration, delaying atheroma plaque formation and aortic valve calcification. Therefore, nestin expression marks cells that regulate inflammatory cell migration during atherosclerosis.

Netrin-1 controls sympathetic arterial innervation.

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion.

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Semaphorin3A, Neuropilin-1, and PlexinA1 are required for lymphatic valve formation.

RATIONALE: The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown. OBJECTIVE: We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation. METHODS AND RESULTS: We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a(-/-) mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a(-/-) mice. CONCLUSIONS: Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.

ALK1 signaling inhibits angiogenesis by cooperating with the Notch pathway.

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-ß/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions.

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B.

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.

Identification and functional analysis of endothelial tip cell-enriched genes.

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.