Activation Of Vascular Endothelial Cells Research Articles

Design and Synthesis of 1,4-Diformyl-Piperazine Ferrostatin-1 Derivatives as Novel Ferroptosis Inhibitors.

The present study focuses on the design and synthesis of novel 1,4-diformyl-piperazine-based ferrostatin-1 (Fer-1) derivatives, and their evaluation against ferroptosis activity. The synthesized compounds demonstrated significant anti-ferroptosis activity in human umbilical vascular endothelial cells (HUVECs), with Compound 24 showing the highest potency. Mechanistic studies revealed that Compound 24 effectively reduced intracellular reactive oxygen species (ROS) levels, mitigated mitochondrial damage, and enhanced glutathione peroxidase 4 (GPX4) expression. Additionally, Compound 24 exhibited improved solubility and plasma stability compared to control compounds, Fer-1 and JHL-12. These findings suggest that 1,4-diformyl-piperazine-based Fer-1 derivatives hold promise as therapeutic agents for ferroptosis-associated cardiovascular diseases.

IPSC-derived megakaryocytes and platelets accelerate wound healing and angiogenesis

BackgroundPlatelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications.MethodsThe growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined.ResultMultiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model.ConclusionOur study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds.

TRNA-derived fragment tRF-30 propels diabetes-induced retinal microvascular complications by regulating STAT3 signaling.

Transfer RNA-derived fragments (tRFs) represent a novel class of non-coding RNA transcripts that possess specific biological functions. However, the involvement of tRFs in retinal microvascular diseases remains poorly understood. In this study, we aimed to reveal whether modulation of tRF-30 expression could attenuate pathological retinal neovascular diseases. Our findings demonstrate a significant upregulation of tRF-30 expression levels in both in vivo models of diabetic retinopathy (DR) and in vitro endothelial sprouting models. Conversely, inhibition of tRF-30 expression suppressed the formation of abnormal neovascularization in the retina in vivo, while reducing the proliferation and migration activity of retinal vascular endothelial cells in vitro. We also found that tRF-30 modulates retinal neovascularization through the tRF-30/TRIB3/signal transducer and activated transcription 3 signaling pathway. Furthermore, we validated a significant upregulation of tRF-30 expression levels in the vitreous humor of DR patients, with high levels of both validity and specificity in diagnostic testing. Collectively, our findings highlight a pro-angiogenic role for tRF-30 in DR. Intervening in the tRF-30 signaling pathway may represent a promising prevention and treatment strategy for retinal angiogenesis.

Impact of angiogenesis inhibitors on inflammatory activation in human vascular endothelial cells

Impact of angiogenesis inhibitors on inflammatory activation in human vascular endothelial cells

RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.

Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.

Promotes M1-polarization and diabetic wound healing using Prussian blue nanozymes

Long-term inflammation and impaired angiogenesis are the main reasons for the difficulty of diabetic wound healing. What to do to effectively promote vascular endothelial cell response and immune cell reprogramming is the key to diabetic skin healing. However, contemporary therapies cannot simultaneously coordinate the promotion of vascular endothelial cells and macrophage polarization, which leads to an increased rate of disability in patients with chronic diabetes. Therefore, we developed a method of repair composed of self-assembling Prussian blue nanoenzymes, which achieved synergistic support for the immune microenvironment, and also contributed to macrophage polarization in the tissue regeneration cycle, and enhanced vascular endothelial cell activity. The template hydrothermal synthesis PB-Zr nanoplatform was prepared and locally applied to wounds to accelerate wound healing through the synergistic effect of reactive oxygen species (ROS). PB-Zr significantly normalized the wound microenvironment, thereby inhibiting ROS production and inflammatory response, which may be because it inhibited the M1 polarization of macrophages in a rat model of wound. PB-Zr treatment significantly promoted the activity of vascular endothelial cells, which better promoted the growth and regeneration of other tissues in the body. The results confirmed the disease microenvironment of PB-Zr-mediated wound therapy and indicated its application in other inflammation-related diseases.

Abstract Or110: Inflammation-induced endothelial cell activation and angiogenic sprouting are downmodulated by ubiquitin-specific peptidase 20

The deubiquitinase ubiquitin-specific peptidase 20 (USP20) reverses lysine-63 ubiquitination of the signaling adaptor TRAF6 and suppresses nuclear-factor-κB (NFκB) activation induced by proatherogenic stimuli in vascular smooth muscle cells (SMCs). Dephosphorylation of USP20 at Ser 334 augments USP20/TRAF6 binding and USP20-mediated TRAF6 deubiquitination, consequently decreasing NFκB signaling. SMC USP20 activity also attenuates atherosclerosis in Ldlr -/- mice. NFκB is a key mediator of vascular endothelial cell (EC) activation and inflammation-driven angiogenesis, which promotes growth and rupture of atherosclerotic plaques. We tested the hypothesis that USP20 attenuates EC NFκB signaling and decreases angiogenesis, with both in vitro and ex vivo models to study the role of USP20 in EC activation and ensuing angiogenesis. We analyzed experimental data by one-way ANOVA followed by Holm-Šídák's multiple comparisons test. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 -/- mice as compared with ECs from congenic wild type (WT) mice (N=3, p<0.001). Assessed by scratch-wound healing and spheroid assays, respectively, migration and angiogenesis of mouse coronary endothelial cells (MCECs) transduced with recombinant adenoviruses was (a) increased by inactive USP20 or phospho-mimetic USP20(S334D) (migration, N=4, P<0.05; angiogenesis, N=6, P<0.01), and (b) decreased by transduction with WT USP20 or phospho-resistant USP20(S334A) (migration, N=4, P< 0.05; angiogenesis, N=6, P<0.01). Additionally, chemical inhibition of NFκB activation with TPCA-1 (which inhibits the upstream kinase IKK-2) attenuated MCEC migration (N=4, P<0.0001) and angiogenesis (N=6, P<0.005), as compared with untreated cells. Angiogenesis assessed by aortic ring sprouting was increased in Usp20 -/- compared with WT specimens (males, N=3, P<0.0001; females, N=2, P<0.0001), and it was also suppressed by TPCA-1 (males, N=2, WT; N=3 Usp20 -/- , P<0.0001; females, N=2 WT; N=3, Usp20 -/- P<0.0005). Angiogenesis was enhanced in aortic rings from USP20(S334D) CRISPR/Cas9 gene-edited mice (males, N=3, P<0.01; females, N=3 WT & N=4, USP20(S334D) P<0.0001), but reduced in aortic rings from USP20(S334A) mice (males, N=3, P<0.05; females, N=3 WT & N=5 USP20(S334A) P<0.0001). We conclude that preserving EC USP20 activity by preventing phosphorylation of USP20 on Ser 334 diminishes endothelial activation and angiogenic sprouting, which can decrease atherosclerosis.

Abstract Tu043: Gut Microbial Metabolite Phenylacetylglutamine Leads to Cardiomyocyte Hypercontractility and Vascular Endothelial Cell Activation

Background: Numerous gut microbial metabolites including phenylacetylglutamine (PAGln) are associated with increased cardiovascular disease risk and mortality. However, little is known about the cellular-specific perturbations by which PAGln may drive cardiovascular dysfunction. Aims: To understand if elevated PAGln leads to cardiovascular dysfunction and what direct cardiac- and vascular-specific pathophysiology is occurring. Methods: Herein, we subject C57Bl/6N male mice to pharmacological increases in circulating PAGln (50mg/kg) or vehicle twice daily for 20 days. We performed echocardiography, invasive hemodynamics, and vascular reactivity assays. We also performed ex vivo cardiomyocyte cell physiology experiments in the presence or absence of PAGln. Moreover, to understand vascular effects we subjected endothelial cells to PAGln and looked at expression of proinflammatory and cell adhesion molecules. Results: We first performed LC-MS/MS analysis demonstrating significant elevation in PAGln levels in multiple tissues compared to vehicle. There was no significant alteration in cardiac structure and function through echocardiographic analysis. Invasive hemodynamic assessment of systemic blood pressure showed no change, however left ventricular filling pressures were significantly elevated in PAGln group. Aortic rings subjected to varying concentrations of acetylcholine in the PAGln-treated animals were significantly impaired compared to vehicle controls. Given the elevated filling pressures and aberrant vascular endothelial relaxation we measured nitrite levels. Nitrite was significantly reduced in the plasma, cardiac, and liver tissue in PAGln group. Cardiomyocyte fractional shortening was significantly increased compared to baseline and equivalent to b-adrenergic agonism. PAGln hypercontractility was not inhibited with adrenergic antagonism. Vascular endothelial cells which were exposed to PAGln had an increase in expression of proinflammatory and cell adhesion molecules versus vehicle. Conclusion: Our data demonstrates that PAGln drives cardiovascular dysfunction through cardiomyocyte hypercontractility and inducing vascular coronary endothelial cell activation in vitro .

Changes of the serum properties and its effect on the endothelial cells restoration in patients with chronic venous disease treated with sulodexide

ObjectiveInflammation and endothelial dysfunction are important venous changes in patients with chronic venous disease (CVD). The use of the venoactive drugs remains an important treatment modality for patients with CVD, reducing the severity of the CVD-related symptoms and swelling but also reducing inflammation and protecting endothelial cells. In this research, the effects of the serum obtained from patients with CVD before and after sulodexide treatment were evaluated for in vivo and in vitro inflammatory markers and endothelial cell function. MethodsInflammatory markers (IL-6, matrix metalloproteinase-9 [MMP-9], vascular cell adhesion molecule-1 [VCAM-1], and von Willebrand factor [vWF]) from the incompetent great saphenous veins (GSVs) and from the systemic venous circulation were studied in 10 patients with CVD (C2s) before and after 2 months of sulodexide (2 × 500 lipasemic units/d) therapy. Serum obtained from the vein blood before and after sulodexide treatment was evaluated for in vitro cultured human umbilical vein endothelial cell function. ResultsThe serum collected from lower leg incompetent GSVs had significantly elevated levels of VCAM-1 (+29%, P < .001) compared with the serum from the systemic circulation. Endothelial cells exposed to the serum from the incompetent lower leg veins of the untreated CVD patients demonstrated higher stimulated synthesis of MMP-9 (+17%, P < .01), as well as increased markers of senescence (prolongation of population doubling time, β-galactosidase activity, and expression of p21 and p53 genes). CVD serum-induced senescent endothelial cells had a higher expression of genes regulating IL-6, MMP-9, VCAM-1, and vWF synthesis. The overall proinflammatory effect on endothelial cells by the serum collected from the incompetent GSVs was stronger as compared with the serum from the systemic circulation. Serum collected from the veins after sulodexide treatment caused lower levels of endothelial cell inflammatory markers as well as respective gene expression than serum obtained at the beginning of the study (before sulodexide treatment). Sulodexide application also reduced the inflammatory secretory activity of the senescent endothelial cells. Sulodexide treatment resulted in the decrease of the majority of the studied inflammatory parameters in both lower limb incompetent vein and systemic blood. ConclusionsIn patients with CVD, there are significant differences between circulating inflammatory markers analyzed from the lower leg incompetent GSV segments compared with the systemic circulation, indicating a higher inflammatory condition in CVD. Treatment with sulodexide reduces the proinflammatory and endothelial cell activation properties of the serum from patients with CVD. Clinical RelevanceThe study documented the significant proinflammatory human vascular endothelial cell activation when exposed to the serum collected from the varicose veins as compared with the serum from the systemic circulation in patients with chronic venous disease (CVD). The inflammatory marker expression, endothelial dysfunction, and endothelial cell senescence transformation can be successfully controlled and downregulated by patients’ exposure to the glycosaminoglycan (sulodexide) treatment. Further studies are needed to confirm if glycosaminoglycan application can prevent further CVD clinical progression due to potential CVD-related pathological processes’ modulation and their downregulation.

Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice.

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.

Allogeneic platelet-rich plasma inhibits ferroptosis in promoting wound repair of type 2 diabetic ulcers

Increasing evidence has revealed the emerging role of ferroptosis in the pathophysiology of type 2 diabetes mellitus (T2DM) and its complications. Platelet-rich plasma (PRP) has been demonstrated to facilitate the healing of T2DM ulcers. However, the mechanism by which PRP repairs T2DM ulcers remains unclear. Here, we sought to investigate the interaction between PRP and ferroptosis in repairing T2DM ulcers. The results showed that the cellular activity, proliferation, and migration of fibroblasts were down-regulated, and the cellular activity and normal function of vascular endothelial cells were impaired in the high glucose environment or under RSL3 conditions (a GSH peroxidase 4 inhibitor and ferroptosis inducer). Additionally, both cells experienced over-activation of multiple forms of reactive oxygen species (ROS) and lipid peroxidation. In the T2DM rat model, we observed a decreased rate of ulcer wound healing, impaired proliferative capacity, diminished vascular regeneration, and marked inflammation and hyperfibrosis. More importantly, there was typical damage to mitochondria, increased levels of iron ions, and consistent alterations in protein expression of ferroptosis-related factors. These factors include cyclooxygenase-2 (COX2), glutathione peroxidase 4 (GPX4), transferrin receptor (TFRC), and Solute Carrier Family 7 Member 11 (SLC7A11), among others. Due to the strong association between ferroptosis and T2DM ulcers, the use of allogeneic platelet-rich plasma (Al-PRP) exhibited physiological effects similar to those of the ferroptosis inhibitor Ferrostatin-1 (Fer-1). In vivo experiments, both drugs inhibited a range of impediments to wound healing caused by T2DM and ameliorated the adverse effects associated with ferroptosis. Moreover, Al-PRP attenuated the impairment of normal cellular function, activation of ROS and lipid peroxidation induced by high glucose or RSL3. These results suggested that ferroptosis was involved in the development of T2DM ulcers, which could be treated with Al-PRP by inhibiting ferroptosis, and inhibition of ferroptosis may be a suitable treatment strategy for T2DM ulcers.

Rat Model of Type 2 Diabetes Mellitus Recapitulates Human Disease in the Anterior Segment of the Eye

Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.

Get two with one: bevacizumab treatment in hereditary hemorrhagic telangiectasia with concomitant cirrhosis

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Weber-Rendu syndrome is a birth defect of the blood vessels that causes telangiectasias and arteriovenous malformations. HHT is a rare, autosomal dominant vascular disorder affecting approximately 1 in 8000 people. This multisystem angiogenic disorder is genetically and phenotypically variable, with the most common symptom being severe and recurrent epistaxis. ALK1, TGF-ß, and VEGF are involved in its pathogenesis. VEGF increases mitotic activity in vascular endothelial cells, leading to uncontrolled angiogenesis and the formation of fragile vessels. Bevacizumab is used in the treatment of HHT by inhibiting VEGF. We present our patient, who developed hepatic encephalopathy due to hemorrhages with diffuse telangiectasias of the skin and tongue due to HHT and achieved an effective response to both conditions with bevacizumab.

Identification of KU-55933 as an anti-atherosclerosis compound by using a hemodynamic-based high-throughput drug screening platform

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 μM), Apicidin (10 μM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations.

BANP Participates in the Chronic Intermittent Hypoxia-Induced Senescence of Vascular Endothelial Cells by Promoting P53 Phosphorylation and Nuclear Retention

Introduction: The objective of this study was to examine the potential induction of senescence in vascular endothelial cells (VECs) by chronic intermittent hypoxia (CIH), a defining characteristic of obstructive sleep apnea (OSA). This investigation seeks to elucidate the underlying mechanisms that contribute to the development of cardiovascular diseases in patients with OSA, with a particular focus on CIH-induced vascular aging. Methods: The BioSpherix-OxyCycler system was used to establish models of CIH in both rats and human umbilical vein endothelial cells (HUVECs). To assess VECs’ senescence, various methods were employed including EdU incorporation assay, cell cycle analysis, senescence-associated β-galactosidase (SA-β-gal) staining, and senescence protein testing. Vascular aging was evaluated through measurements of carotid-femoral pulse wave velocity, intima-media thickness, and Ki67 immunohistochemical staining. In order to identify the molecular mechanisms associated with CIH-induced senescence in VECs, a bioinformatics study was conducted utilizing the Gene Expression Omnibus database. Results: Under conditions of CIH, HUVECs exhibited inhibited proliferation, arrested cell cycle, increased activity of SA-β-gal, and elevated expression levels of p53 and p21 compared to HUVECs under normoxic conditions. Similarly, rats exposed to CIH displayed increased carotid-femoral pulse wave velocity, intima-media thickness, vascular permeability, and SA-β-gal activity in VECs, along with decreased expression of arterial Ki67. BTG3-associated protein (BANP) was found to be highly expressed in CIH-induced VECs. Furthermore, the overexpression of BANP resulted in the senescence of VECs, along with elevated levels of p53 phosphorylation and nuclear localization. Conclusions: These findings demonstrate that CIH can induce VECs senescence and contribute to vascular aging. Additionally, BANP can induce VECs senescence by promoting p53 phosphorylation and nuclear retention. These discoveries offer novel insights into the increased cardiovascular risk associated with OSA, thereby presenting new possibilities for therapeutic intervention.

CD62E- and ROS-Responsive ETS Improves Cartilage Repair by Inhibiting Endothelial Cell Activation through OPA1-Mediated Mitochondrial Homeostasis.

Background: In the environment of cartilage injury, the activation of vascular endothelial cell (VEC), marked with excessive CD62E and reactive oxygen species (ROS), can affect the formation of hyaluronic cartilage. Therefore, we developed a CD62E- and ROS-responsive drug delivery system using E-selectin binding peptide, Thioketal, and silk fibroin (ETS) to achieve targeted delivery and controlled release of Clematis triterpenoid saponins (CS) against activated VEC, and thus promote cartilage regeneration. Methods: We prepared and characterized ETS/CS and verified their CD62E- and ROS-responsive properties invitro. We investigated the effect and underlying mechanism of ETS/CS on inhibiting VEC activation and promoting chondrogenic differentiation of bone marrow stromal cells (BMSCs). We also analyzed the effect of ETS/CS on suppressing the activated VEC-macrophage inflammatory cascade invitro. Additionally, we constructed a rat knee cartilage defect model and administered ETS/CS combined with BMSC-containing hydrogels. We detected the cartilage differentiation, the level of VEC activation and macrophage in the new tissue, and synovial tissue. Results: ETS/CS was able to interact with VEC and inhibit VEC activation through the carried CS. Coculture experiments verified ETS/CS promoted chondrogenic differentiation of BMSCs by inhibiting the activated VEC-induced inflammatory cascade of macrophages via OPA1-mediated mitochondrial homeostasis. In the rat knee cartilage defect model, ETS/CS reduced VEC activation, migration, angiogenesis in new tissues, inhibited macrophage infiltration and inflammation, promoted chondrogenic differentiation of BMSCs in the defective areas. Conclusions: CD62E- and ROS-responsive ETS/CS promoted cartilage repair by inhibiting VEC activation and macrophage inflammation and promoting BMSC chondrogenesis. Therefore, it is a promising therapeutic strategy to promote articular cartilage repair.

Baicalin inhibited PANX-1/P2Y6 signaling pathway activation in porcine aortic vascular endothelial cells infected by Glaesserella parasuis

Glaesserella parasuis can induce endothelial barrier damage in piglets, although the mechanism by which this pathogen triggers inflammatory damage remains unclear. Baicalin possesses anti-inflammatory and anti-oxidant activities. However, whether baicalin can relieve endothelial barrier damage caused by Glaesserella parasuis infection has not yet been studied. Hence, we evaluated the ability of baicalin to counteract the changes induced by Glaesserella parasuis in porcine aortic vascular endothelial cells. The results showed that Glaesserella parasuis could upregulate the expression of pannexin 1 channel protein and promote the release of adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate in porcine aortic vascular endothelial cells. The expression level of purinergic receptor P2Y6 was upregulated in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. In addition, Glaesserella parasuis could activate phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells. Baicalin could inhibit pannexin 1 channel protein expression, reduce adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate release, and attenuate the expression level of P2Y6 in porcine aortic vascular endothelial cells induced by Glaesserella parasuis. Baicalin could also reduce the activation of phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. Our study report that Glaesserella parasuis could promote pannexin 1 channel protein expression, induce nucleosides substance release, and P2Y6 expression in porcine aortic vascular endothelial cells and baicalin could inhibit the expression levels of pannexin 1, nucleosides substance, and P2Y6 in the porcine aortic vascular endothelial cells induced by Glaesserella parasuis, which might be served as some targets for treatment of inflammation disease caused by Glaesserella parasuis.

Predictive Biomarker Analysis from the GBT021601 Survival Study in Townes Sickle Mice

Introduction : Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene resulting in sickle hemoglobin (HbS). Red blood cell (RBC) deoxygenation leads to HbS polymerization, RBC sickling and subsequent chronic hemolysis, anemia, vascular endothelial cell activation, vaso-occlusion, and ischemia. SCD is associated with extensive morbidity and markedly reduced survival. GBT021601 is an investigational, second-generation HbS polymerization inhibitor that reduces RBC sickling and anemia, and improves both O 2 delivery and consumption in chronically treated Townes Sickle Cell mice that harbor 2 HbS alleles (SS). Objectives of this 2-part study were to (1) characterize the effects of GBT021601 administered ad libitum on compound exposure and pharmacodynamics (PD), (2) examine the impact of long-term (&amp;gt;24 weeks) treatment with GBT021601 on % reticulocytes and overall survival (OS) of Townes SS mice, and (3) assess the utility of early changes in % reticulocytes as a predictive biomarker of survival in Townes SS mice treated with GBT021601. Methods: In Part 1, 24 male Townes SS mice 8-10 weeks old were split into 3 cohorts (n=8/group) and for 3 weeks were fed standard chow containing 0% (control), 0.05% (low dose), or 0.2% (high dose) of GBT021601. Body weight and food consumption were measured weekly. Mice were bled daily on days 10-21 to determine RBC half-life and on days 10 and 21 for analysis of compound exposure. On day 22 mice were euthanized. Whole blood was collected for analyses of compound exposure, % reticulocytes, Hb levels, RBC numbers, RBC half-life, and RBC deformability. In Part 2, 5-week-old Townes mice (AA and SS genotype; 50% male) were fed either standard chow (AA [n=57] and SS mice [n=54]) or chow containing 0.05% or 0.2% of GBT021601 (SS mice only; n=55 each). Body weight and food consumption were measured weekly. Blood was collected at 1, 14, 31, 43, 55, and 67 weeks of treatment for compound exposure analysis and % reticulocytes. Mice were euthanized when the humane end-point criteria were met or after 72 weeks of treatment, and whole blood was collected for compound exposure and % reticulocyte analyses. Results: GBT021601 in chow was well tolerated during both study parts; food consumption and body weight were unaffected. Outcomes in Part 1 confirmed the same compound exposure/PD trends as mice orally gavaged with GBT021601 as observed by Dufu et al. (Br J Haematol 2023); Hb levels, RBC numbers, and RBC half-life increased, RBC deformability improved, and % reticulocytes decreased. Long-term dosing of GBT021601 in the chow of Townes SS mice revealed similar compound exposure and % reticulocytes as our 3-week dosing study (Part 1) and Dufu et al. In Part 2, Townes SS mice fed GBT021601 chow, achieving compound exposures after 1 week of dosing as depicted in Figure 1, had a longer median survival vs control Townes SS mice (60 vs 56 weeks). In control Townes SS mice, % reticulocytes increased during the first 14 weeks of the study and then plateaued, whereas in Townes SS mice fed GBT021601 there was a dose-dependent sustained reduction in % reticulocytes compared with control Townes SS mice. Reductions in % reticulocytes after 1 week of dosing were greater in Townes SS mice achieving a higher compound exposure (Figure 1), a trend sustained after 14 and 31 weeks of treatment. OS increased in Townes SS mice of the same sex achieving a ≤30% reduction in % reticulocytes vs Townes SS mice with % reticulocytes &amp;gt;30% after 1 week of treatment; P=0.00048 (Figure 2). Multivariate survival analysis, including % reticulocytes at 1 week (categorical variable of ≤30% or &amp;gt;30%) and sex as predictors confirmed significant association of &amp;gt;30% reticulocytes with reduced survival (hazard ratio=1.48, P=0.027 using Cox proportional hazard). Conclusions:Longer-term GBT021601 treatment as part of chow was well tolerated by Townes SS mice, and compound exposure was comparable with studies of a shorter duration of treatment. Early compound exposure was predictive of a long-term survival benefit in Townes SS mice. Early reductions in % reticulocytes were more likely in Townes SS mice with a high GBT021601 exposure and were predictive of a long-term survival benefit. Overall, our findings support the use of GBT021601 to reduce % reticulocytes and improve OS in Townes SS mice and suggest the clinical relevance of early reductions in % reticulocytes as a biomarker to predict long-term survival.

Investigation of the biological activity of hydroxyapatite on vascular endothelial cells

ObjectiveThis study aims to explore the impact of nano-hydroxyapatite (na-HA) and micron-hydroxyapatite (mi-HA) on human umbilical vein endothelial cells (HUVEC) using in vitro experiments, assessing their influence on cellular biological activity. These findings offer crucial experimental data for informing the development of more vascularized tissue-engineered bone constructs. MethodsWe employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of various concentrations of both HA extracts on HUVEC metabolic activity post 48, 72, and 96 h of treatment. Transwell experiments were conducted to evaluate the influence of HA extract on HUVEC migratory capabilities. The cell proliferation activity was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, elucidating the impact of varying concentrations of both HA extracts on cell proliferation. Lumen formation experiments were conducted to assess the capacity of HA-treated HUVECs to form lumen-like structures. The Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the impact of HA extract on vascular endothelial growth factor (VEGF) secretion by HUVECs. Western blotting (WB) was utilized to analyze alterations in the expression levels of PI3K/Akt signaling pathway-related proteins following HA extract treatment of cells. ResultsAt extract concentrations of 100 g/L and 12.5 g/L, both the mi-HA and na-HA groups demonstrated suppression of cell metabolic activity, migration, and proliferation. Conversely, at 25 g/L, increased cell metabolic activity and proliferative activity were observed. Lumen formation experiments demonstrated that both HA extracts at 100 g/L concentration facilitated lumen formation, with the na-HA group at 25 g/L concentration displaying a more pronounced impact on lumen formation. The ELISA results indicated a notable reduction in VEGF secretion within the mi-HA group at a concentration of 100 g/L. WB experiments revealed that within the na-HA group, treatment of HUVECs with 25 g/L and 12.5 g/L extract concentrations led to upregulation of PI3K and Akt protein expression, while at 100 g/L concentration, Akt protein expression decreased. In the mi-HA group, intracellular expression of both PI3K and Akt proteins exhibited reduction. ConclusionHydroxyapatite extract at both high and low concentrations impacts the biological activity of vascular endothelial cells, with the potential mechanism of action involving the PI3K/Akt signaling pathway.

Apohemoglobin-Hpatoglobin Promotes Blood Flow in Sickle Cell Disease Mice

In sickle cell disease (SCD), a single change in the β-globin gene coding sequence results in the production of hemoglobin S (HbS). This HbS polymerizes when deoxygenated, causing red blood cell (RBC) sickling, hemolysis, anemia, reduced blood flow in small vessels, activation of vascular endothelial cells, vaso-occlusion, and ischemia. These abnormalities lead to various clinical complications, including fatigue, painful vaso-occlusive crises (VOC), reduced quality of life, organ damage, and early death. The management of SCD involves supportive care, pain management, blood transfusions, and disease-modifying therapies, all aimed at alleviating symptoms and improving the overall quality of life for affected individuals. We propose a new therapy that uses a biologic called apohemoglobin-haptoglobin (apoHb-Hp), which scavenges free circulating Hb and heme. This study examined the efficacy of apoHb-Hp in reducing vaso-occlusion in a mouse model of sickle cell disease (SCD) using transgenic mice expressing HbSS-Towne (sickle cell mice) and compared them to mice expressing normal Hb, HbAA-Towne. A single dose (10 mg/kg) of normal saline (control), Haptoglobin (Hp), or apoHb-Hp was administered before the experiment, and twenty-four subcutaneous venules were selected and mapped after baseline selection. The same vessels were re-examined for stasis at 2, 8, 24, and 48 hours, and the percentage of stasis was calculated. The results demonstrated that microvascular stasis occurs spontaneously in the Towns transgenic mice model but can be reduced by a single dose of the apoHb-Hp complex. In another set of experiments, mice were pretreated with the apoHb-Hp complex or Hp and then challenged with hypoxia-reoxygenation (hypoxia of 10% oxygen for one hour) and followed for 48 hours. Hypoxia induced microvascular stasis, also known as vaso-occlusion, which increased post-hypoxia and slowly disappeared over time in Towns mice. The study showed that pre-treatment with the apoHb-Hp complex or Hp improved the rates of recovery from vaso-occlusive crises. A single dose of the apoHb-Hp complex also reduced vascular stasis. In the last study, we administered Hb mixed with either Ringer's lactate, apoHb-Hp complex, or Hp to Towns mice fitted with the dorsal window. We knew that exogenous Hb could cause vascular stasis in Towns mice, so we aimed to investigate the effectiveness of the apoHb-Hp complex compared to Hp in preventing Hb-induced vascular stasis. The Hb infusion led to maximal stasis within an hour in the control group. Therefore, microvascular stasis occurred one hour after infusing Hb alone or with equimolar apoHb-Hp complex and Hp. Our results showed that mice co-infused with Hb + apoHb-Hp complex or Hb + Hp had significantly lower stasis levels compared to mice infused with Hb alone. Surprisingly, the apoHb-Hp complex was more effective than Hp alone in reducing Hb-induced stasis. These findings provide insight into the underlying mechanisms of the apoHb-Hp complex and demonstrate its efficacy in reducing vaso-occlusion. Further studies are necessary to optimize the therapeutic use of the apoHb-Hp complex.