Cultures Of Human Endothelial Cells Research Articles

Use of modified human hemangioma tissue cultures and human umbilical vein endothelial cell cultures to gain mechanistic insights into imiquimod treatment for infantile hemangioma.

Infantile hemangiomas (IH) are a common entity encountered by dermatologists, otolaryngologists, and other surgeons. Oral propranolol is a mainstay of treatment for IH and is well-tolerated, though propranolol-refractory IH and other drug-related adverse events are documented and can limit its usage. There are few in vitro testing systems for putative treatment agents. To address this, we modified a tissue culture system for human hemangioma treatment testing to evaluate the treatment impact of the immune modifier, imiquimod. Human umbilical vein endothelial cells (HUVEC) and hemangioma cultures were treated with several concentrations of imiquimod followed by MTT assays, reporter gene assays, PCR, ELISA, and Western blotting for IL-8, VEGF, Cyclin D1, and IFNα and immunohistochemistry for Cyclin D1 and Ki-67. HUVEC showed acute decreases in IL-8, VEGF, and Cyclin D1 promoter activity and increases in IFNα mRNA after imiquimod treatment. Hemangioma samples showed no change in Ki-67 or Cyclin D1 staining after treatment with imiquimod after 27 d, with significantly increased IL-8 and VEGF. From this preliminary analysis, we discerned that hemangioma tissues can be grown in tissue culture and used for drug treatment studies. We also conclude acute and chronic modulation of cell cycle, angiogenesis factors, and immunostimulatory conditions may be associated with imiquimod mechanisms of action in hemangioma involution.

H2S-Eluting Hydrogels Promote In Vitro Angiogenesis and Augment In Vivo Ischemic Wound Revascularization

Ischemic wounds are frequently encountered in clinical practice and may be related to ischemia secondary to diabetes, peripheral artery disease and other chronic conditions. Angiogenesis is critical to the resolution of ischemia. Hydrogen sulfide (H2S) is now recognized as an important factor in this process. H2S donors NaHS and GYY4137 were incorporated into the photosensitive polymer hydrogel gelatin methacrylate and evaluated. Human umbilical vein endothelial cell (HUVEC) culture was used to quantify toxicity and angiogenesis. Sprague Dawley rats were subjected to ischemic myocutaneous flap wound creation with and without application of H2S-eluting hydrogels. Tissue perfusion during wound healing was quantified using laser speckle contrast imaging, and gene and protein expression for VEGF were evaluated. Vascular density was assessed by CD31 immunohistochemistry. Successful incorporation of sulfide compounds was confirmed by scanning electron microscopy with energy-dispersive X-ray analysis, and under physiologic conditions, detectable H2S was present for up to 14 days by high-performance liquid chromatography. HUVECs exposed to hydrogels did not demonstrate excess cytotoxicity or apoptosis. A two-fold increase in angiogenic tube formation was observed in HUVECs exposed to H2S-eluting hydrogels. Rat ischemic flap wounds demonstrated greater perfusion at 14 days, and there was greater vascularity of healed wounds compared to untreated animals. A nearly two-fold increase in VEGF mRNA and a four-fold increase in VEGF protein expression were present in wounds from treated animals. Local-regional administration of H2S represents a novel potential therapeutic strategy to promote angiogenesis and improve wound healing after tissue injury or as a result of ischemic disease.

A 3D in vitro model of biphasic calcium phosphate (BCP) scaffold combined with human osteoblasts, osteoclasts, and endothelial cells as a platform to mimic the oral microenvironment for tissue regeneration

Objectives: This study aimed to develop an innovative 3D in vitro model based on the biphasic calcium phosphate (BCP) scaffold combined with human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells to evaluate its effects on bone and vascular cells behavior. Methods: To this end, an optimized mixture of hydroxyapatite (HA) and β-tricalcium phosphate (TCP) with a weight ratio of 30/70 was employed to develop a BCP scaffold using the computer-aided design (CAD) approach. The BCP scaffold was combined with primary cultures of hOBs, hOCs and human umbilical vein endothelial cells (HUVECs). Results: Morphometric analyses using scanning electron microscopy (SEM) and X-ray micro-computed tomography, along with biomechanical testing, revealed that BCP scaffold exhibited a regular 3D structure with large interconnected internal pores (700 µm) and high mechanical strength. In terms of biological behavior, after 14 days of tri-culture with hOBs, hMCs and HUVECs, SEM, immunofluorescence, and histological analyses showed that all cell types were viable and adhered well to the entire surface of the scaffold. Interestingly, SEM and energy-dispersive X-ray spectroscopy analyses also revealed on the BCP scaffold the presence of mineralized matrix crystals of Ca, P, O and C within a tissue-like cell layer produced by the interaction of the three cell types. Conclusions: Data confirmed the high performance of the BCP scaffold through biomechanical studies. Notably, for the first time, this study demonstrated the feasibility of combining BCP scaffold with hOBs, hOCs, and HUVEC, which remained viable and maintained their native phenotypes, creating also tissue-like cell layer. Clinical significanceAlthough further investigation is needed, these results underscore the potential to develop a 3D in vitro model that mimics the oral microenvironment, which could be valuable for BTE approaches in in vivo studies.

Phosphatidylserine-blocking nanoparticles inhibit thrombosis without increased bleeding in mice

BackgroundPhosphatidylserine (PS) is a procoagulant phospholipid enriched on surfaces of activated vascular cells including platelets, endothelium, monocytes, and microvesicles. As a molecular driver of thrombosis accessible to drug blockade, PS is an attractive pharmacologic target for modulating thrombogenesis, with potentially reduced bleeding risk compared to anticoagulant and antiplatelet therapies. ObjectivesTest antithrombotic capabilities of a liposomal formulation, Zn-dipicolylamine cyanine-3[22,22]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (molar ratio, 3:97), designated as DPAL, which we previously described binds selectively to PS-enriched cell surfaces, compared with effects on bleeding, in mouse models. MethodsPS-dependent DPAL binding to human and murine platelets was tested in vitro. Thrombosis and bleeding after DPAL intravenous administration were tested in C57Bl/6J mice following FeCl3 carotid arterial injury and tail tip amputation, respectively. Incorporation in hemostatic clots was investigated in the cremaster muscle laser injury model. Toxicity was tested by direct exposure to human endothelial cell cultures. ResultsDPAL bound agonist-stimulated, PS-positive human and murine platelets, blocked by Annexin V or Ano6 deletion, which ablate PS exposure. DPAL prolonged prothrombin time, but did not prevent thrombin-induced fibrinogen receptor activation or aggregation, nor alter blood cell counts including platelets. Following arteriolar laser injury, DPAL bound wound surfaces and edges without destabilizing plugs. DPAL dose-dependently blocked FeCl3-induced arterial thrombosis but did not substantially increase bleeding, or induce endothelial cell death. ConclusionDPAL reduces thrombogenesis with minimal effects on bleeding in mouse models via selective binding to PS. DPAL may support novel approaches to modulate pathogenic thrombin generation with improved safety profiles in multiple contexts.

Human Chorionic Membrane-derived Tunable Hydrogels for Vascular Tissue Engineering Strategies.

One of the foremost targets in the advancement of biomaterials to engineer vascularized tissues is not only to replicate the composition of the intended tissue but also to create thicker structures incorporating a vascular network for adequate nutrients and oxygen supply. For the first time, to the best of current knowledge, a clinically relevant biomaterial is developed, demonstrating that hydrogels made from the human decellularized extracellular matrix can exhibit robust mechanical properties (in the kPa range) and angiogenic capabilities simultaneously. These properties enable the culture and organization of human umbilical vein endothelial cells into tubular structures, maintaining their integrity for 14 days in vitro without the need for additional polymers or angiogenesis-related factors. This is achieved by repurposing the placenta chorionic membrane (CM), a medical waste with an exceptional biochemical composition, into a valuable resource for bioengineering purposes. After decellularization, the CM underwent chemical modification with methacryloyl groups, giving rise to methacrylated CM (CMMA). CMMA preserved key proteins, as well as glycosaminoglycans. The resulting hydrogels rapidly photopolymerize and have enhanced strength and customizable mechanical properties. Furthermore, they demonstrate angio-vasculogenic competence in vitro and in vivo, holding significant promise as a humanized platform for the engineering of vascularized tissues.

SARS-CoV-2 S Protein Reduces Cytoprotective Defenses and Promotes Human Endothelial Cell Senescence.

Premature vascular aging and endothelial cell senescence are major risk factors for cardiovascular diseases and atherothrombotic disturbances, which are main complications of both acute and long COVID-19. The S protein of SARS-CoV2, which acts as the receptor binding protein for the viral infection, is able to induce endothelial cells inflammation and it has been found as an isolated element in the circulation and in human tissues reservoirs months after infection. Here, we investigated whether the S protein is able to directly induce endothelial cell senescence and deciphered some of the mechanisms involved. In primary cultures of human umbilical vein endothelial cells (HUVEC), SARS-CoV-2 S protein enhanced in a concentration-dependent manner the cellular content of senescence and DNA damage response markers (senescence-associated-β galactosidase, γH2AX), as well as growth-arrest effectors (p53, p21, p16). In parallel, the S protein reduced the availability of cytoprotective proteins, such as the anti-aging protein klotho, Nrf2 or heme oxygenase-1, and caused functional harm by impairing ex vivo endothelial-dependent vasorelaxation in murine microvessels. These effects were prevented by the pharmacological inhibition of the NLRP3 inflammasome with MCC950. Furthermore, the supplementation with either recombinant klotho or angiotensin-(1-7), equally protected against the pro-senescence, pro-inflammatory and pro-oxidant action of the S protein. Globally, this study proposes novel mechanisms of disease in the context of COVID-19 and its vascular sequelae and provides pharmacological clues in order to prevent such complications.

Biocompatibility Testing of UV-Curable Polydimethylsiloxane for Human Umbilical Vein Endothelial Cell Culture on-a-Chip.

Polydimethylsiloxane (PDMS) is extensively used to fabricate biocompatible microfluidic systems due to its favorable properties for cell culture. Recently, ultraviolet-curable PDMS (UV-PDMS) has shown potential for enhancing manufacturing processes and final optical quality while retaining the benefits of traditional thermally cured PDMS. This study investigates the biocompatibility of UV-PDMS under static and flow conditions using human umbilical vein endothelial cells (HUVECs). UV-PDMS samples were treated with oxygen plasma and boiling deionized water to assess potential improvements in cell behavior compared with untreated samples. We evaluated HUVECs adhesion, growth, morphology, and viability in static cultures and microchannels fabricated with UV-PDMS to test their resistance to flow conditions. Our results confirmed the biocompatibility of UV-PDMS for HUVECs culture. Moreover, plasma-oxygen-treated UV-PDMS substrates exhibited superior cell growth and adhesion compared to untreated UV-PDMS. This enhancement enabled HUVECs to maintain their morphology and viability under flow conditions in UV-PDMS microchannels. Additionally, UV-PDMS demonstrated improved optical quality and more efficient handling and processing, characterized by shorter curing times and simplified procedures utilizing UV light compared to traditional PDMS.

Bio-Inspired Screwed Conduits from the Microfluidic Rope-Coiling Effect for Microvessels and Bronchioles

Tubular microfibers have recently attracted extensive interest for applications in tissue engineering. However, the fabrication of tubular fibers with intricate hierarchical structures remains a major challenge. Here, we present a novel one-step microfluidic spinning method to generate bio-inspired screwed conduits (BSCs). Based on the microfluidic rope-coiling effect, a viscous hydrogel precursor is first curved into a helix stream in the channel, and then consecutively packed as a hollow structured stream and gelated into a screwed conduit (SC) via ionic and covalent crosslinking. By taking advantage of the excellent fluid-controlling ability of microfluidics, various tubes with diverse structures are fabricated via simple control over fluid velocities and multiple microfluidic device designs. The perfusability and permeability results, as well as the encapsulation and culture of human umbilical vein endothelial cells (HUVECs), human pulmonary alveolar epithelial cells (HPAs), and myogenic cells (C2C12), demonstrate that these SCs have good perfusability and permeability and the ability to induce the formation of functional biostructures. These features support the uniqueness and potential applications of these BSCs as biomimetic blood vessels and bronchiole tissues in combination with tissue microstructures, with likely application possibilities in biomedical engineering.

Alleviation of Angiotensin II-Induced Vascular Endothelial Cell Injury Through Long Non-coding RNA TUG1 Inhibition.

Hypertension damages endothelial cells, causing vascular remodelling. It is caused by Ang II-induced endothelial cell (EC) destruction. The long noncoding RNA (lncRNAs) are emerging regulators of endothelium homeostasis. Injured endothelium expresses lncRNA taurine-upregulated gene 1 (TUG1), which may mediate endothelial cell damage, proliferation, apoptosis, and autophagy and contribute to cardiovascular disease. However, uncertainty surrounds the function of lncRNA TUG1, on arterial endothelium cell damage. This research aimed to investigate the role and mechanism of lncRNA TUG1 in vascular endothelial cell injury. A microarray analysis of lncRNA human gene expression was used to identify differentially expressed lncRNAs in human umbilical vein endothelial cell (HUVEC) cultures. The viability, apoptosis, and migration of Ang II-treated HUVECs were then evaluated. In order to investigate the role of lncRNA TUG1 in hypertension, qRT-PCR, western blotting, and RNA-FISH were used to examine the expression of TUG1 in SHR mice. Ang II-activated HUVECs and SHR rats' abdominal aortas highly express the lncRNA TUG1. LncRNA TUG1 knockdown in HUVECs could increase cell viability, reduce apoptosis, and produce inflammatory factors. In SHR rat abdominal aortas, lncRNA TUG1 knockdown promoted proliferation and inhibited apoptosis. HE spotting showed that lncRNA TUG1 knockdown improved SHR rats' abdominal aorta shape. lncRNA TUG1 knockdown promotes miR-9- 5p, which inhibits CXCR4 following transcription. The lncRNA TUG1/miR-9-5p/CXCR4 axis and vascular cell injury were also examined. MiR-9-5p silencing or CXCR4 overexpression lowered cell survival, apoptosis, and lncRNA TUG1-induced IL-6 and NO expression. lncRNA TUG1 suppression could reduce Ang II-induced endothelial cell damage by regulating and targeting miR-9-5p to limit CXCR4 expression and open new vascular disease research pathways.

Endothelial peroxiredoxin-4 is indispensable for blood–brain barrier integrity and long-term functional recovery after ischemic stroke

The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury.

Dehydroepiandrosterone promotes ovarian angiogenesis and improves ovarian function in a rat model of premature ovarian insufficiency by up-regulating HIF-1α/VEGF signalling

Research questionWhat impact does dehydroepiandrosterone (DHEA) have on ovarian angiogenesis and function in a rat model of with premature ovarian insufficiency (POI), and what are the potential mechanisms of action? DesignDHEA was added to a culture of human microvascular endothelial cells (HMEC-1) to investigate its effects on cell proliferation, migration and tube formation. A rat model of POI was established by intraperitoneal injection of cyclophosphamide, followed by continuous oral administration of DHEA or vehicle for 28 days. Ovarian angiogenesis, follicular growth and granulosa cell survival in ovarian tissues were assessed through haematoxylin and eosin staining, immunohistochemistry and TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL). The effect of DHEA on the fertility of rats with POI was evaluated in pregnant animals. The expression levels of characteristic genes and proteins in the hypoxia-inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) pathway was determined using quantitative reverse transcription PCR and western blotting. ResultsIn-vitro experiments revealed that DHEA stimulated the proliferation, migration and tube formation of HMEC-1. In in-vivo studies, DHEA treatment improved the disruption of the oestrous cycle and hormone imbalances in POI rats. Key genes in the HIF-1α/VEGF pathway exhibited up-regulated expression, promoting ovarian angiogenesis in POI rats, and enhancing follicular development and granulosa cell survival, thereby restoring fertility in rats. ConclusionsDHEA can potentially restore ovarian function in rats with cyclophosphamide-induced POI by up-regulating HIF-1α/VEGF signalling, which promotes the growth of blood vessels in the ovaries.

SH2 domain protein E and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis.

Colony-forming endothelial cells – candidate culture for tissue vascular engineering: the gene and proteomic profile

Aim. To validate ECFC culture as a candidate culture for vascular tissue engineering using comparative analysis of the proteomic and gene expression profiles in comparison with cultures of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC).Materials and Methods. ECFC culture was obtained by cultivating peripheral blood mononuclear cells of patients with coronary artery disease. Commercial HCAECs produced by Cell Applications, and HUVECs cultured according to the modified protocol of Jaffe were used as controls.The cells were lysed with TRIzol, and total RNA was isolated using a Purelink RNA Micro Scale Kit with concomitant DNase treatment. Next, rRNA depletion was carried out, followed by the creation of DNA libraries. DNA libraries were quantified using quantitative polymerase chain reaction on a CFX96 Touch Bio-Rad amplifier. DNA libraries were equimolarly mixed and sequenced on HiSeq 2000 (Illumina) with a paired-end reads of 2x125 nucleotides.Conventional western blotting was performed using pan-endothelial markers CD31, vWF, VEG-FR2/KDR, marker of endothelial progenitor cells CD34, markers of epithelial-mesenchymal transition Snail and Slug, and markers of endothelial specification: arterial HEY2, venous COUP-TFII and lymphatic LYVE1, VEGFR2. Dot blotting against 55 angiogenesis-related proteins was performed using Proteome Profiler Human Angiogenesis Array Kit in accordance with the manufacturer's protocol.Results. ECFC overexpresses markers of all three endothelial lineages (KDR, VWF, CD34, NRP2, FLT4 and LYVE1 compared to HCAEC; NOTCH4, DLL2) and LYVE1 compared to HUVEC. Proteomic profiling indicated ECFC as an intermediate population between HCAEC and HU-VEC in term of the expression of HEY2, LYVE1, VEGFR3, Snail and Slug. 261 DEGs were detected between ECFC and HUVEC, and 470 DEGs between ECFC and HCAEC.Conclusion. The gene expression profile of endothelial colony-forming cells corresponds to mature endothelial cells and indicates ECFC as an intermediate population between HCAEC and HUVEC. ECFC culture can be recommended for tissue vascular engineering.

Development of tissue-engineered vascular grafts from decellularized parsley stems.

Cardiovascular diseases are mostly associated with narrowing or blockage of blood vessels, and it is the most common cause of death worldwide. The use of vascular grafts is a promising approach to bypass or replace the blocked vessels for long-term treatment. Although autologous arteries or veins are the most preferred tissue sources for vascular bypass, the limited presence and poor quality of autologous vessels necessitate seeking alternative biomaterials. Recently, synthetic grafts have gained attention as an alternative to autologous grafts. However, the high failure rate of synthetic grafts has been reported primarily due to thrombosis, atherosclerosis, intimal hyperplasia, or infection. Thrombosis, the main reason for failure upon implantation, is associated with damage or absence of endothelial cell lining in the vascular graft's luminal surface. To overcome this, tissue-engineered vascular grafts (TEVGs) have come into prominence. Alongside the well-established scaffold manufacturing techniques, decellularized plant-based constructs have recently gained significant importance and are an emerging field in tissue engineering and regenerative medicine. Accordingly, in this study, we demonstrated the fabrication of tubular scaffolds from decellularized parsley stems and recellularized them with human endothelial cells to be used as a potential TEVG. Our results suggested that the native plant DNA was successfully removed, and soft tubular biomaterials were successfully manufactured via the chemical decellularization of the parsley stems. The decellularized parsley stems showed suitable mechanical and biological properties to be used as a TEVG material, and they provided a suitable environment for the culture of human endothelial cells to attach and create a pseudo endothelium prior to implantation. This study is the first one to demonstrate the potential of the parsley stems to be used as a potential TEVG biomaterial.

Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that exvivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Exvivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Preparation and evaluation of a suspension of human corneal endothelial cells isolated from the eyes of cadaveric donors for transplantation in an <em>ex vivo</em> experiment

Background. According to the World Health Organization, corneal diseases are one of the major causes of blindness globally. Endothelial dystrophy is one of the etiological factors leading to corneal diseases. The corneal endothelium is a monolayer of cells with virtually no mitotic activity. When the density of corneal endothelial cells falls below a critical threshold, the endothelium loses its ability to regulate corneal stromal hydration. This leads to corneal clouding and, consequently, to reduced visual acuity and quality of life of the patient. In this regard, various keratoplasty methods are widely used in clinical practice. Today, it is technically possible to transplant all corneal layers via penetrating keratoplasty, and to transplant the posterior epithelium via layer-bylayer keratoplasty. These surgical approaches are now widely used in everyday practice, but they require the use of scarce material – cadaveric donor corneas, from which grafts for the above-mentioned operations are formed in the conditions of an eye bank. In this regard, protocols for obtaining human corneal endothelial cell (HCEC) culture for subsequent transplantation have been proposed in recent years. However, the use of such approaches in Russia is limited by the law. The aim of this study was to experimentally justify the possibility of transplanting uncultured endothelial cells, isolated from cadaveric human corneas. Materials and methods. The first stage of the work consisted of obtaining a suspension of endothelial cells from cadaveric donor corneas and studying it; at the second stage, the transplantation effectiveness of the resulting cell suspension was assessed in an ex vivo experiment. Results. The cell phenotype after transplantation by the proposed method had high viability and preservation. Conclusions. The presented results suggest that phenotype and adhesion ability are preserved, and that the cell suspension has a high level of viability under adequate loss of endothelial cells during transplantation in the ex vivo experiment.

An effective method for culturing functional human corneal endothelial cells using a xenogeneic free culture medium

Endothelial dysfunction is a leading cause of corneal blindness in developed countries and the only available treatment is the endothelial transplantation. However, the limited availability of suitable donors remains a significant challenge, driving the exploration of alternative regenerative therapies. Advanced Therapy Medicinal Products show promise but must adhere to strict regulations that prohibit the use of animal-derived substances. This study investigates a novel culture methodology using Plasma Rich in Growth Factors (PRGF) as the only source of growth factors for primary cultures of human corneal endothelial cells (CECs). CECs were obtained from discarded corneas or endothelial rings and cultured in two different media: one supplemented with xenogeneic factors and other xenogeneic-free, using PRGF. Comprehensive characterization through immunofluorescence, morphological analyses, trans-endothelial electrical resistance measurements, RNA-seq, and qPCR was conducted on the two groups. Results demonstrate that CECs cultured in the xenogeneic-free medium exhibit comparable gene expression, morphology, and functionality to those cultured in the xenogeneic medium. Notably, PRGF-expanded CECs share 46.9% of the gene expression profile with native endothelium and express all studied endothelial markers. In conclusion, PRGF provides an effective source of xenogeneic-free growth factors for the culture of CECs from discarded corneal tissue. Further studies will be necessary to demonstrate the applicability of these cultures to cell therapies that make clinical translation possible.

Abstract 18413: Therapeutic Genome Editing of Endothelial FABP4 by Endothelium-Targeted Nanoparticle Delivery of the CRISPR/Cas9 System Inhibits Pulmonary Arterial Hypertension in Mice and Rats

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling. The molecular mechanisms of obliterative pulmonary vascular remodeling remain elusive. Hypothesis: Therapeutic genome editing of endothelial Fabp4 will knockout FABP4 expression selectively in endothelial cells and inhibit PAH in mice and rats. Methods: Employing the newly developed endothelial cell (EC)-targeted nanoparticle delivery technology, plasmid DNA expressing CRISPR/Cas9 controlled by Cdh5 promoter and Fabp4 -specific guide RNAs driven by U6 promoter was administered i.v. to adult SD rats and Egln1 Tie2Cre mice which exhibit severe PAH and obliterative pulmonary vascular remodeling. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, right ventricular systolic pressure (RVSP), ratio of right ventricular versus left ventricular plus septum (RV/LV+S) and pulmonary vascular remodeling were determined. Pharmacological FABP4 inhibitor was also employed to treat MCT rats as a comparison. Similarly, the pulmonary hypertensive phenotype in genome-edited Egln1 Tie2Cre mice was also characterized. Results: Nanoparticle delivery of the all-in-one CRISPR rCdh5 plasmid DNA knockout Fabp4 selectively in endothelial cells in adult rats and Egln1 Tie2Cre mice leading to reduced RVSP, RV hypertrophy and pulmonary vascular remodeling in MCT rats and also in Egln1 Tie2Cre mice. Obliterative pulmonary vascular remodeling seen in Egln1 Tie2Cre mice was inhibited and the mortality rate in endothelial Fabp4-deficient Egln1 Tie2Cre mice was reduced. Treatment of FABP4 inhibitor also inhibits PAH in MCT rats. We also observed a marked increase of FABP4 expression in endothelial cells of lung samples of IPAH patients. Overexpression of FABP4 in primary cultures of human lung microvascular endothelial cells resulted in a 70% increase of cell proliferation. Conclusions: These findings demonstrate endothelial FABP4 is a promising target for PAH therapy. EC-targeted nanoparticle delivery of the genome editing system may represent an effective and safer gene therapy approach for PAH treatment.

Nerve pathology of microangiopathy and thromboinflammation in hereditary transthyretin amyloidosis.

Despite amyloid deposition as a hallmark of hereditary transthyretin amyloidosis (ATTRv) with polyneuropathy, this pathology could not completely account for nerve degeneration. ATTRv patients frequently have vasomotor symptoms, but microangiopathy hypothesis in ATTRv was not systemically clarified. This study examined the vascular pathology of sural nerves in ATTRv patients with transthyretin (TTR) mutation of p.Ala117Ser (TTR-A97S), focusing on morphometry and patterns of molecular expression in relation to nerve degeneration. We further applied human microvascular endothelial cell (HMEC-1) culture to examine the direct effect of TTR-A97S protein on endothelial cells. In ATTRv nerves, there was characteristic microangiopathy compared to controls: increased vessel wall thickness and decreased luminal area; both were correlated with the reduction of myelinated fiber density. Among the components of vascular wall, the area of collagen IV in ATTRv nerves was larger than that of controls. This finding was validated in a cell model of HMEC-1 culture in which the expression of collagen IV was upregulated after exposure to TTR-A97S. Apoptosis contributed to the endothelial cell degeneration of microvasculatures in ATTRv endoneurium. ATTRv showed prothrombotic status with intravascular fibrin deposition, which was correlated with (1) increased tissue factor and coagulation factor XIIIA and (2) reduced tissue plasminogen activator. This cascade led to intravascular thrombin deposition, which was colocalized with upregulated p-selectin and thrombomodulin, accompanied by complement deposition and macrophages infiltration, indicating thromboinflammation in ATTRv. Microangiopathy with thromboinflammation is characteristic of advanced-stage ATTRv nerves, which provides an add-on mechanism and therapeutic target for nerve degeneration.