Search Results (3,242)

Vascular basement membranes (BMs), composed of laminins, collagen IV, fibronectin, and perlecan, are secreted by endothelial cells, pericytes, smooth muscle cells (SMCs), and astrocytes. In the brain, amyloid beta (Aβ) is eliminated along cerebrovascular BMs of capillaries and arteries as intramural periarterial drainage (IPAD). Ageing modifies vascular BMs, impairing IPAD and leading to Aβ deposition as cerebral amyloid angiopathy. To better understand the molecular determinants of IPAD in ageing, we quantified the relative abundance of BMs secreted by human-derived cerebral endothelial cells, pericytes, brain vascular SMCs, and astrocytes in vitro. We then assessed BM protein levels in SMCs under hypercapnia (8% CO₂) as a model of vascular ageing, with and without Aβ exposure. Of the four cell types, we found SMCs secreted the highest levels of fibronectin, laminin, and perlecan, whilst pericytes secreted the highest levels of collagen IV. Hypercapnia increased the expression of collagen IV and fibronectin in SMCs but decreased the expression of laminin. The expression of perlecan increased under hypercapnia, but only in the presence of Aβ. This work highlights the varying compositions of vascular BMs and the dynamic differential responses of SMCs to Aβ and hypercapnia, helping to elucidate the age-related changes that impair IPAD in cerebral vessels. Full article

Background/Objectives: The low permeability of the blood-brain barrier (BBB) represents a significant challenge to effective systemic chemotherapy for primary and metastatic brain cancers. Kinin receptors play a crucial role in modulating BBB permeability, and their agonist analogs have been explored in preclinical animal models to enhance drug delivery to the brain. In this study, we investigated whether des-Arg⁹-bradykinin (DBK), a physiological agonist of kinin B₁ receptor (B1R), acts as a brain drug delivery adjuvant by promoting the transient opening of the BBB. Methods: Human brain microvascular endothelial cells (HBMECs) were treated with DBK in the culture medium and in conditioned media from glioblastoma cell lines, namely T98G (CMT98G) and U87MG (CMU87). Immunofluorescence, RT-qPCR, in-cell Western assay, and proximity ligation assay (PLA) were performed to analyze BBB components, kinin receptors and TLR4, a receptor associated with the kinin pathway and inflammation. The effect of DBK on enhancing paracellular molecule transport was evaluated using Evans blue dye (EB) quantification in a cell culture insert assay and in an in vivo model, where mice with and without brain tumors were treated with DBK. To assess the functional impact of the transient BBB opening induced by DBK, the chemotherapeutic drug doxorubicin (DOX) was administered. Results: Treatment with DBK facilitates the presence of EB in the brain parenchyma by transiently disrupting the BBB, as further evidenced by the increased paracellular passage of the dye in an in vitro assay. B1R activation by DBK induces transient BBB opening lasting less than 48 h, enhancing the bioavailability of the DOX within the brain parenchyma and glioma tumor mass. The interaction between B1R and TLR4 is disrupted by the secreted factors released by glioblastoma cells, as conditioned media from T98G and U87 reduce TLR4 staining in endothelial cells without affecting B1R expression. Conclusions: These results further support the potential of B1R activation as a strategy to enhance targeted drug delivery to the brain. Full article

Background/Objective: A clinical need exists for more effective therapeutics and sustained drug delivery systems to promote ocular surface healing. This study tested the hypothesis that a novel biodegradable, thermoresponsive hydrogel loaded with the human recombinant (rh)MG53 protein, which we have demonstrated to promote corneal healing without fibrosis, would exhibit safety and biocompatibility in vitro and in vivo. Methods: Hydrogel optimization was performed based on varying concentrations of poloxamer 407, poloxamer 188, and hydroxypropyl methylcellulose. Hydrogels were characterized and potential toxicity was evaluated in vitro in cultured corneal epithelium, fibroblasts, and endothelium. In vivo safety and tolerability were assessed in mice and hydrogels were used to evaluate corneal healing following alkali injury. Results: The optimized hydrogel formulation did not result in any detrimental changes to the corneal cells and released functional rhMG53 protein for at least 24 h. In vivo rhMG53-loaded hydrogels improved re-epithelialization, reduced stromal opacification and vascularization, and promoted corneal nerve density. Mechanistically, rhMG53 reduced vascular endothelial cell migration and tube formation by inhibiting pSTAT3 signaling. Conclusions: Taken together, our poloxamer-based thermoresponsive hydrogel effectively released rhMG53 protein and enhanced multiple corneal healing outcomes. Full article

Hair loss, a prevalent condition affecting individuals across various demographics, is associated with hormonal imbalances, oxidative stress, inflammation, and environmental factors. This study evaluated the anti-hair loss potential of the water-soluble fraction of Rhus semialata gall extract (WRGE) and its primary component, Penta-O-Galloyl-β-D-Glucose (PGG), through both in vitro and clinical studies. WRGE was obtained using a standardized extraction process, and PGG was identified via HPLC-DAD and HRESIMS/MS techniques. Human dermal papilla cells (HDPCs) are specialized fibroblasts that can regulate the hair growth cycle and hair follicle growth. HDPCs are widely used in research focused on anti-hair loss. In this study, the anti-hair loss effects of WRGE and PGG on HDPCs were confirmed. WRGE and PGG enhance cell proliferation in HDPCs. These results are associated with the activation of the Wnt/β-catenin signaling pathway and the upregulation of hair growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and fibroblast growth factor (FGF). Furthermore, WRGE and PGG significantly inhibited dihydrotestosterone (DHT)-mediated DKK-1 secretion and H₂O₂-medicated cytotoxicity. Clinical trials further validated these results, demonstrating significant improvements in hair density and visual hair appearance scores in participants treated with WRGE compared to a placebo group. These results collectively suggest that WRGE and PGG may serve as promising natural agents for the prevention and treatment of hair loss by targeting multiple biological pathways, including the regulation of hair growth factors, oxidative stress, and hormonal imbalances. Full article

Patients with end-stage renal disease (ESRD) are at increased risk of cardiovascular disease (CVD), such as myocardial infarction (MI). Uremic toxins and endothelial dysfunction are central to this process. In this exploratory study, we used the Affymetrix GeneChip microarray to investigate the gene expression profile in uremic serum-induced human coronary arterial endothelial cells (HCAECs) from ESRD patients with and without MI (UWI and UWOI groups) as an approach to its underlying mechanism. We also explored which pathways are involved in this process. We found 100 differentially expressed genes (DEGs) among the conditions of interest by supervised principal component analysis and hierarchical cluster analysis. The expressions of four major DEGs were validated by quantitative RT-PCR. Pathway analysis and molecular network were used to analyze the interaction and expression patterns. Ten pathways were identified as the main enriched metabolic pathways according to the transcriptome profiling analysis, which were, among others, positive regulation of inflammatory response, positive regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) cascade, cardiac muscle cell development, highlighting positive regulation of mitogen-activated protein kinase (MAPK) activity (p = 0.00016). Up- and down-regulation of genes from HCAECs exposed to uremic serum could contribute to increased endothelial dysfunction and CVD in ESRD patients. Our study suggests that inflammation and the ERK-MAPK pathway are highly enriched in kidney disease patients with MI, suggesting their role in ESRD pathology. Further studies and approaches based on MAPK pathway interfering strategies are needed to confirm these data. Full article

Salt-sensitive hypertension (SSH) is closely associated with arterial inflammation, yet its molecular mechanisms remain unclear. In this study, we utilized deoxycorticosterone acetate (DOCA)-salt-induced hypertensive mice, which exhibited elevated blood pressure and significant arterial inflammation. Single-cell RNA sequencing (scRNA-seq) identified interferon regulatory factor 5 (IRF5) and its downstream targets, signal transducer and activator of transcription (STAT), as key regulators of these inflammatory changes. In vivo, IRF5 levels were significantly elevated in the DOCA group, while STAT1 and STAT2 protein levels were comparable to those in the normal salt group. However, nuclear levels of phosphorylated STAT1 (pSTAT1) and phosphorylated STAT2 (pSTAT2) were markedly higher in the DOCA group. Furthermore, scRNA-seq analysis showed increased IRF5 expression in endothelial cells (ECs) in both human and mouse aorta samples. In vitro, IRF5 knockdown in artery ECs led to a reduction in nuclear pSTAT1 and pSTAT2 expression. These results suggest that IRF5 promotes STAT1 and STAT2 phosphorylation, enabling their nuclear translocation. Additionally, RNA sequencing indicated a positive correlation between endothelial cell-specific molecule 1 (ESM1) and STAT1/STAT2. Using the UCSC and JASPAR databases, we identified multiple binding sites for the STAT1::STAT2 dimer on the ESM1 promoter. Luciferase reporter assays revealed enhanced ESM1 transcription following pSTAT1::pSTAT2 binding, and pinpoint potential binding sites. Chromatin Immunoprecipitation Quantitative PCR (ChIP-qPCR) further confirmed the specific binding sites between the pSTAT1::pSTAT2 dimer and the ESM1 promoter. These findings highlight the critical role of the IRF5-pSTAT1::pSTAT2-ESM1 pathway in the pathogenesis of SSH and suggest potential therapeutic targets. Full article

SARS-CoV-2 has demonstrated a remarkable capacity for immune evasion. While initial studies focused on the Wuhan variant and adaptive immunity, later emerging strains such as Omicron exhibit mutations that may alter their immune-modulatory properties. We performed a comprehensive review of immune evasion mechanisms associated with SARS-CoV-2 viral proteins to focus on the evolutionary dynamics of immune modulation. We systematically analyzed and compared the impact of all currently known Wuhan and Omicron SARS-CoV-2 proteins on type I interferon (IFN) responses using a dual-luciferase reporter assay carrying an interferon-inducible promoter. Results revealed that Nsp1, Nsp5, Nsp14, and ORF6 are potent type I IFN inhibitors conserved across Wuhan and Omicron strains. Notably, we identified strain-specific differences, with Nsp6 and Spike proteins exhibiting enhanced IFN suppression in Omicron, whereas the Envelope protein largely retained this function. To extend these findings, we investigated selected proteins in primary human endothelial cells and also observed strain-specific differences in immune response with higher type I IFN response in cells expressing the Wuhan strain variant, suggesting that Omicron’s adaptational mutations may contribute to a damped type I IFN response in the course of the pandemic’s trajectory. Full article

Hypercholesterolemia causes atherosclerosis by inducing immune cell migration and chronic inflammation in arterial walls. Recent single-cell analyses reveal the presence of lipid-enriched foamy macrophages, as well as other macrophage subtypes, neutrophils, T cells, and B cells, in atherosclerotic plaques in both animal models and humans. These cells interact with each other and other cells, including non-immune cells such as endothelial cells and smooth muscle cells. They thereby regulate metabolic, inflammatory, phagocytic, and cell death processes, thus affecting the progression and stability of atherosclerotic plaques. The nuclear receptors liver X receptor (LXR)α and LXRβ are transcription factors that are activated by oxysterols and regulate lipid metabolism and immune responses. LXRs regulate cholesterol homeostasis by controlling cholesterol’s transport, absorption, synthesis, and breakdown in the liver and intestine. LXRs are also highly expressed in tissue-resident and monocyte-derived macrophages and other immune cells, including both myeloid cells and lymphocytes, and they regulate both innate and adaptive immune responses. Interestingly, LXRs have immunosuppressive and immunoregulatory functions that are cell-type-dependent. In animal models of atherosclerosis, LXRs have been shown to be involved in both progression and regression phases. The pharmacological activation of LXR enhances cholesterol efflux from macrophages and promotes atherosclerosis progression. Deleting LXR in immune cells, especially myeloid cells, accelerates atherosclerosis by increasing monocyte migration, macrophage proliferation and activation, and neutrophil extracellular traps (NETs); furthermore, the deletion of hematopoietic LXRs impairs the regression of atherosclerotic plaques. Therefore, LXRs in immune cells may be a potent therapeutic target for atherosclerosis. Full article

Black chokeberry (BCK), known as Aronia melanocarpa (Michx.) Elliott, has been employed for various purposes throughout history, being exploited both for its nutritional properties (functional foods, beverages, food preservatives, and natural food colorants) and for its therapeutic benefits (including cardiovascular and metabolic settings). This paper presents the first report on the identification of inorganic elements in three standardized BCK extracts: frozen berries (FrozArs), dried berries (DryArs), and evaporated juice (EvArJ). Additionally, the antiproliferative and pro-apoptotic effects of these extracts on human pulmonary artery endothelial cells (HPAECs) were evaluated. Concentrations ranging from 1 μg/mL to 10 μg/mL were tested. Inorganic element analysis revealed detectable levels of metals, including aluminum (Al), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Notably, cadmium was found in very low amounts (0.026 μg/g in the FrozArs), while iron was the most abundant element in the juice (597.665 μg/g). MTT assays demonstrated that all three extracts exhibited antiproliferative activity against HPAECs. Cell cycle analysis revealed a decrease in the G2/M phase for all extracts, along with an appearance of the sub-G0 phase at the highest concentration tested. The DryAr extract also slightly reduced the number of cells in the G0-G1 phase. Annexin V/PI staining indicated a mild increase in the percentage of necrotic cells associated with the DryAr extract. The potential implications of these findings are significant, particularly for those interested in the health effects of dietary supplements. Full article

Background: We studied the effects of human blood coagulation on antioxidant activity and the cellular secretion of immunoregulatory molecules in vitro. Methods: Reactive oxygen species (ROS) activity and cytokine content were determined in plasma and serum blood samples incubated with lipopolysaccharide (LPS) for 3 h or 18 h. Results: Coagulation process significantly decreased ROS activity induced by LPS in blood samples from healthy donors. Human serum was found to have significantly higher antioxidant activity than plasma. Blood coagulation markedly reduced LPS-induced secretion of TNF-α by cells, without significantly affecting the secretion of interleukin-1 (IL-1), IL-6, IL-8, or C-reactive protein (CRP). Blood clotting led to an increase in LPS-induced release of vascular endothelial growth factor (VEGF) by blood cells. A significant increase in procalcitonin levels was also observed in serum samples. Conclusions: Blood clotting enhances the antioxidant and anti-inflammatory functions of immunoreactive blood cells. Full article

Background: TOTUM-854 is a patented plant extract blend characterized by its components that have previously been described for their potential health benefits in limiting hypertension onset. However, most of the literature data remain descriptive regarding the mode of action at the cellular level, especially in humans, and further investigations are required for optimized therapeutic strategies. Methods: We first demonstrated in an L-NAME mouse model that TOTUM-854 supports the prevention of hypertension in vitro and in vivo. Then, we designed an ex vivo clinical innovative approach considering the circulating metabolites produced by the digestive tract upon TOTUM-854 ingestion in humans. Human serum was collected in healthy volunteers before and after the acute intake of 3.71 g of TOTUM-854. The bioavailability of circulating metabolites was confirmed and characterized by UPLC-MS. Human serum containing TOTUM-854-derived metabolites was further processed for incubation with human endothelial cells (HUVECs), in the absence or presence of palmitate (200 µM). Results: HUVEC protection against lipotoxicity was characterized by (1) decreased ACE-1 activity (−32% p < 0.0001); (2) the inhibition of oxidative stress with decreased ROS (−12% observed by DCFDA and DHE fluorescent microscopy) and decreased Nox2 gene expression (−6.7 fold change vs. palmitate, p < 0.01); and (3) the inhibition of an inflammatory response, with a decrease in IL-1β release (−37% compared to palmitate, p < 0.001) and decreased MCP-1 and VCAM-1 gene expression (−93% p < 0.001 and −77% p < 0.001, respectively). Conclusions: Overall, this study provides insightful data regarding the protective role of TOTUM-854 in human endothelial cells. Using an innovative clinical ex vivo approach, our data support the role of TOTUM-854 circulating metabolites in vascular protection in humans. Full article

The venom of Macrovipera lebetinus obtusa (MLO) has remarkable properties that are hard to overlook. This venom’s described 38 protein components work synergistically, forming complexes that greatly enhance their combined effectiveness. Previous studies have shown that both crude venom and one of its components, obtustatin, can reduce sarcoma tumors by 50% and 30%, respectively. Obtustatin, a member of the short disintegrin family, inhibits the angiogenic activity of α1β1 integrin, the adhesive receptor of collagen IV. However, the mechanisms of the greater efficacy of the crude venom compared to its isolated components remain unclear. To investigate this, we propose an experimental work to explore the activity of certain low-molecular-weight components of MLO venom. Our in vitro tests on fibrosarcoma (HT-1080) cells using six venom fractions revealed cytotoxic fractions, which, through mass spectrometry, were identified as containing protein classes such as dimeric and short disintegrins, acidic phospholipase A2, and serine proteinases. Notably, these fractions exhibited minimal toxicity to human dermal microvascular endothelial (HDEC) cells, suggesting their potential as a promising candidate for oncotherapy in the future. Full article

Current in vitro models fail to recapitulate specific physiological properties of the human blood–brain barrier (BBB); hence the need for a reliable platform to study central nervous system diseases and drug permeability. To mimic the normally tight blood–brain interface, primary human endothelial cells (HAECs) and primary human astrocytes (A) were grown in a confined space of the physical scaffold created by gelatin methacrylate (GelMA) hydrogel to allow optimal astrocyte–endothelial cell direct/indirect interaction. Evidence for a physiologically relevant BBB was established by assessing the expression of tight junction markers conferring the barrier function, and by measuring biophysical attributes using the trans-endothelial electrical resistance (TEER) and the Evans blue albumin (EBA) permeability assay. An HAEC+A three-dimensional (3D) co-culture was associated with 12-fold higher claudin-5 (CLDN5) and cadherin-1 (CDH1 or Epithelial [E]-cadherin) transcriptional levels than two-dimensional (2D) models. This model conferred the highest TEER (45 Ω·cm²) in 3D HAEC+A, which value was 30 Ω·cm² in 2D (p < 0.01) and 25 Ω·cm² in 3D HAEC cultures (p < 0.001). Functionally, in 3D HAEC+A co-cultures, higher TEER resulted in 10-fold and 7-fold lower EBA permeability at 120 min, in HAECs alone or in to 2D co-cultures (p < 0.01). The established human primary cell model has acquired features mimicking the human BBB in vitro, and is now poised to be tested for the permeability of the BBB to pharmacological agents, parasites, cells (such as brain-tropic cancer cell metastasis) and any mechanisms that might involve traversing the BBB. Full article

Porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), a porcine herpesvirus, has been shown to significantly reduce the survival time of porcine xenotransplants in non-human primates. The virus was detected in all the examined organs of baboons transplanted with PCMV/PRV-positive organs and it was also transmitted to the first human recipient of a pig heart, contributing to the patient’s death. PCMV/PRV induces consumptive coagulopathy and thrombocytopenia in xenotransplant recipients. Initial studies in baboons revealed that the virus triggered increased release of tumor necrosis factor α (TNFα) and interleukin 6 (IL-6), along with elevated levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) complexes. Since there is no evidence that PCMV/PRV infects primate cells, including human cells, the virus appears to directly interact with immune and endothelial cells, disrupting cytokine signaling and coagulation pathways. The highest viral load was detected in the explanted pig heart, suggesting active replication at this site. Additionally, cells expressing PCMV/PRV proteins were identified in all the examined baboon organs, where pig cells were also found. Since PCMV/PRV affects only xenotransplant recipients and not healthy humans, this condition should be classified as a xenozoonosis. Interestingly, antibodies against human herpesvirus 6 (HHV-6) cross-react with PCMV/PRV and may contribute to protection against infection in humans. Further research is needed to uncover the molecular mechanisms underlying this xenozoonotic disease. Full article

Background: Hypertension is a major risk factor for cerebrovascular diseases due to its damaging effects on the blood–brain barrier (BBB) and associated pathologies. Oxidative stress-induced endothelial damage plays a critical role in BBB disruption, potentially leading to cognitive impairment and neurodegeneration. In this study, we investigated the protective effects of two essential trace elements, zinc (Zn) and selenium (Se), against oxidative stress in human brain endothelial cells (HBCE5i) exposed to hypertensive shear stress. Using an innovative millifluidic system (LiveBox2), which allows for the precise simulation of continuous flow conditions, we replicated the hemodynamic forces associated with hypertension. Methods: Cells were treated with ZnCl₂ (5–50 µM) or Na₂SeO₃ (50–500 nM) at concentrations selected based on previous studies and confirmed by cytotoxicity assays. Results: Our results demonstrated that shear stress significantly altered the localization of the tight junction protein zonula occludens-1 (ZO-1) and induced the nuclear translocation of the transcription factor NRF2, a hallmark of oxidative stress. Importantly, treatment with 10 µM ZnCl₂ preserved ZO-1 membrane localization and prevented NRF2 translocation, as confirmed by quantitative image analysis. In contrast, Na₂SeO₃ did not provide comparable protection, although modest improvements in ZO-1 localization were observed in some replicates. Discussion: We discuss potential reasons for selenium’s limited efficacy, including differences in bioavailability and cellular uptake. Our findings underscore zinc’s promising role as a neurovascular protector and suggest that further investigation into more complex in vitro models and in vivo studies is warranted. Full article