Endothelial Healing Research Articles

369-P: Wound Repair by P53 Silenced Human Endothelial Cell-Derived Conditioned Media, In Vitro

Introduction: Diabetes is associated with impaired wound healing. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature human endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment and p53 gene silencing prevents cellular senescence and helps vascular regeneration. Our goal was to test whether cell conditioned media obtained from p53 silenced human cells can also improve cellular regeneration. Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control. To collect condition media, different human cells such as HEK, human CD14+ve cells and HUVEC (human umbilical vein endothelial cells) were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% BSA (with no FBS and growth factors) for only 24 h and the collected supernatant was concentrated 10-fold to obtain the conditioned media (CM). Endothelial regeneration or wound healing was tested using endothelial wound healing assay kit (from Cell Biolabs # CBA-120-T). Results: We compared CM obtained from null and p53sh in HEK, CD14+ve and HUVEC cells on human endothelial cells. Proliferation capacity of the endothelial cells was greater with p53sh CM compared to null and non-transduced CM. The CM obtained from the untreated and null samples did not show increased proliferation in any of the 3 kinds of cells tested. The CM from p53sh HUVEC was more effective than CM from other cells. We are analyzing the CM (exosome and non-exosome fraction) for identification of specific secretory proteins. Conclusion: P53 silencing appears to improve HUVEC paracrine factors and this particular modality of cell free therapy may have therapeutic role in treating diabetes complications and even aging. Disclosure D. K. Rivera: None. S. Nandula: None. S. Sen: None. Funding American Heart Association

Novel Supreme Drug-Eluting Stents With Early Synchronized Antiproliferative Drug Delivery to Inhibit Smooth Muscle Cell Proliferation After Drug-Eluting Stents Implantation in Coronary Artery Disease: Results of the PIONEER III Randomized Clinical Trial.

Accelerated endothelial healing after targeted antiproliferative drug delivery may limit the long-term inflammatory response of drug-eluting stents (DESs). The novel Supreme DES is designed to synchronize early drug delivery within 4 to 6 weeks of implantation, leaving behind a prohealing permanent base layer. Whether the Supreme DES is safe and effective in the short term and can improve long-term clinical outcomes is not known. In an international, 2:1 randomized, single-blind trial, we compared treatment with Supreme DES to durable polymer everolimus-eluting stents (DP-EES) in patients with acute and chronic coronary syndromes. The primary end point was target lesion failure-a composite of cardiac death, target vessel myocardial infarction, or clinically driven target lesion revascularization. The trial was designed to demonstrate noninferiority (margin of 3.58%) of the Supreme DES at 12 months compared with DP-EES (URL: https://www.clinicaltrials.gov; Unique identifier: NCT03168776). From October 2017 to July 2019, a total of 1629 patients were randomly assigned (2:1) to the Supreme DES (N=1086) or DP-EES (N=543). At 12 months, target lesion failure occurred in 57 of 1057 patients (5.4%) in the Supreme DES group and in 27 of 532 patients (5.1%) in the DP-EES group (absolute risk difference, 0.32% [95% CI, -1.87 to 2.5]; Pnoninferiority=0.002]. There were no significant differences in rates of device success, clinically driven target lesion revascularization, or stent thrombosis at 12 months, and the safety composite of cardiovascular death and target vessel myocardial infarction was 3.5% versus 4.6% (hazard ratio, 0.76 [95% CI, 0.46-1.25]) with Supreme DES compared with DP-EES, although rates of combined clinically and non-clinically driven target lesion revascularization at 12 months were higher with Supreme DES. Among patients with acute and chronic coronary syndromes undergoing percutaneous coronary intervention, the Supreme DES proved to be noninferior to the standard DP-EES. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03168776.

EG Coated Stents Exhibit Enhanced Endothelial Wound Healing Characteristics

PurposeDespite their widespread use, a significant fraction of coronary stents suffer from in-stent restenosis and stent thrombosis. Stent deployment induces extensive injury to the vascular endothelium. Rapid endothelial wound closure is essential for the success of a stenting procedure. A recent study has demonstrated that the BuMA Supreme® sirolimus-eluting stent exhibits particularly attractive strut coverage characteristics. A unique feature of this stent is the presence of a thin brush layer of poly-butyl methacrylate (PBMA), covalently bonded to the stent’s cobalt-chromium frame via electro-grafting (eG™). The present study aimed to determine whether the PBMA coating has an effect on endothelial cell wound healing and stent strut coverage.MethodsWe used an in vitro coronary artery model whose wall consisted of an annular collagen hydrogel and whose luminal surface was lined with a monolayer of endothelial cells. Mechanical wounding of the endothelial lining was preformed prior to deployment of a bare cobalt-chromium stent either with or without the PBMA layer. The migration of fluorescently labeled endothelial cells was monitored automatically over a period of 48 h to determine endothelial wound healing rates.ResultsQuantitative assessment of endothelial wound healing rates within the simulated arterial model is achievable using automated image analysis. Wound healing is significantly faster (44% faster at 48 h) for stents with the PBMA eG Coating™ compared to bare metal stents.ConclusionThe PBMA eG Coating™ has the effect of promoting endothelial wound healing. Future studies will focus on elucidating the mechanistic basis of this observation.

Role of β 3 ‐Adrenergic Receptors in Rat Aortic Endothelial Cell Function

Long-term success of percutaneous trans-luminal coronary angioplasty (PTCA) is limited by the frequent occurrence of restenosis, resulted from vascular smooth muscle cell (VSMC) proliferation and migration due to the procedure-induced endothelial damage. Speeding the healing of the endothelial layer has been considered as one of the major approaches to reduce the risk of restenosis. Basic fibroblast growth factor (bFGF), a major growth factor released after injury, regulates endothelial cell proliferation and migration and promotes endothelial layer healing. Our previous results revealed that β3-adrenergic receptor (β3-ARs) expression is upregulated in newly formed intima in injured blood vessels and activation of β3-ARs enhanced bFGF-induced VSMC proliferation and migration. However, the role of β3-ARs in endothelial function, specifically in injured arteries, is uncertain. The purpose of this study is to determine if activation of endothelial cell β3-ARs potentiates bFGF-induced endothelial cell proliferation and/or migration by using rat aortic endothelial cells. Cell proliferation was determined by measuring viable cell numbers using Resazurin. Cell migration will be quantified by a monolayer wound healing assay in the presence of a cell proliferation inhibitor, hydroxyurea. A dose-dependent inhibition of cell proliferation was performed to optimize the concentration of hydroxyurea in cell migration experiments. Our results revealed that pretreatment of endothelial cells with a selective β3-AR agonist, CL316,243, enhanced bFGF-induced vascular endothelial cell proliferation. We also found that 5μM of hydroxyurea completely inhibited cell proliferation induced by growth factors. In conclusion, our data suggest that activation of β3-ARs may speed the healing of endothelial layer by promoting cell proliferation and that including 5μM of hydroxyurea in cell migration experiments will allow us to measure the cell motility accurately and ensure that proliferation does not take place.

Inhibition of SGLT2 Rescues Bone Marrow Cell Traffic for Vascular Repair: Role of Glucose Control and Ketogenesis.

The mechanisms by which sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve cardiovascular outcomes in people with diabetes are incompletely understood. Recent studies show that SGLT2i may increase the levels of circulating cells with vascular regenerative capacity, at least in part by lowering glycemia. In this study, we used mice with streptozotocin-induced diabetes treated with the SGLT2i dapagliflozin at a dose that reduced glucose levels by 20%. Dapagliflozin improved the diabetes-associated defect of hematopoietic stem cell mobilization after stimulation with granulocyte colony-stimulating factor. Dapagliflozin rescued the traffic of bone marrow (BM)-derived cells to injured carotid arteries and improved endothelial healing in diabetic mice. Defective homing of CD49d+ granulocytes was causally linked with impaired endothelial repair and was reversed by dapagliflozin. The effects of dapagliflozin were mimicked by a similar extent of glucose reduction achieved with insulin therapy and by a ketone drink that artificially elevated β-hydroxybutyrate. Inhibition of endothelial repair by resident cells using the CXCR4 antagonist AMD3100 did not abolish the vascular effect of dapagliflozin, indirectly supporting that endothelial healing by dapagliflozin was mediated by recruitment of circulating cells. In summary, we show that dapagliflozin improved the traffic of BM-derived hematopoietic cells to the site of vascular injury, providing a hitherto unappreciated mechanism of vascular protection.

SDF-1\u03b1 gene-activated collagen scaffold enhances provasculogenic response in a coculture of human endothelial cells with human adipose-derived stromal cells

Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1α GAS) could be used to enhance functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1α GAS with a gene-free scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the VE-cadherin. On the 14th day, the coculture on the SDF-1α GAS showed enhanced maturation than its gene-free equivalent through the elevation of provasculogenic genes (SDF-1α—7.4-fold, CXCR4—1.5-fold, eNOS—1.5-fold). Furthermore, we also found that the coculture on SDF-1α GAS secretes bioactive factors that significantly (p < 0.01) enhanced human Schwann cells’ clustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1α GAS could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between endothelial cells and ADSCs.

Multiple roles of FGF10 in the regulation of corneal endothelial wound healing

Corneal endothelial dysfunction usually induces corneal haze and oedema, which seriously affect visual function. The main therapeutic strategy for this condition is corneal transplantation, but the use of this strategy is limited by the shortage of healthy donor corneas. Compared with corneal transplantation, drug intervention is less invasive and more accessible; thus, finding an effective pharmaceutical alternative for cornea transplantation is critical for the treatment of corneal endothelial dysfunction. In this study, we established a rabbit scratch model to investigate the effect of fibroblast growth factor 10 (FGF10) on corneal endothelial wound healing. Results showed that FGF10 injection accelerated the recovery of corneal transparency and increased the protein expression levels of ZO1, Na+/K+-ATPase and AQP-1. Moreover, FGF10 significantly inhibited the expression levels of endothelial-to-mesenchymal transition proteins and reduced the expression levels of the proinflammatory factors IL-1β and TNF-α in the anterior chamber aqueous humour. FGF10 also enhanced the Na+/K+-ATPase activity by enhancing mitochondrial function as a result of its direct interaction with its conjugate receptor. Thus, FGF10 could be a new pharmaceutical preparation as treatment for corneal endothelial dysfunction.

Rho kinase (ROCK) inhibitors in the management of corneal endothelial disease.

Rho kinase (ROCK) inhibitors are growing increasingly relevant in ophthalmology, and the goal of this review is to summarize their mechanisms of action and potential applications in the subspecialties of glaucoma, retina, and cornea. We will focus specifically on corneal endothelial wound healing, for which ROCK inhibition demonstrates particular promise. ROCK inhibition has been shown to promote corneal endothelial cell proliferation, increase intercellular adhesion, and suppress apoptosis. Topical ROCK inhibitor treatment has exhibited potential use in Fuchs endothelial dystrophy, corneal edema from acute surgical trauma and other etiologies, and tissue engineering therapy for the endothelial disease. Ripasudil and netarsudil, the two ROCK inhibitors available for ophthalmic use, are generally very well tolerated with mild and transient local side effects. ROCK inhibitors are revolutionizing the subspecialty of cornea, and further research is needed to compare long-term outcomes of ROCK inhibitor therapy to those of conventional endothelial keratoplasty, including visual acuity and endothelial cell density. Other possible avenues include the use of ROCK inhibitors to prolong corneal graft survival, and early data appears promising.

Syndecan-1 Overexpressing Mesothelioma Cells Inhibit Proliferation, Wound Healing, and Tube Formation of Endothelial Cells.

Simple SummaryThe transmembrane proteoglycan syndecan-1 (SDC-1) is an important mediator of cell-matrix interactions. The heparan sulfate side-chains of SDC-1 can bind to a multitude of growth factors, cytokines, and chemokines, thereby regulating a plethora of physiological and pathological processes, including angiogenesis. The extracellular region of SDC-1 can be released from the cell surface by the action of sheddases including matrix metalloproteinase-7 and 9, resulting in a soluble protein that is still active and can act as a competitive activator or inhibitor of the cell surface receptor. Accelerated shedding and loss of cell surface SDC-1 is associated with epithelial to mesenchymal transition (EMT) and achievement of a more invasive phenotype in malignant mesothelioma (MM). Transfection with SDC-1 reverts the morphology in epithelioid direction and inhibits the proliferation and migration of MM cells. This study aimed to investigate the role of SDC-1 in angiogenesis. We demonstrate that overexpression and silencing of SDC-1 alters the secretion of angiogenic proteins in MM cells. Upon SDC-1 overexpression, several factors collectively inhibit the proliferation, wound closure, and tube formation of endothelial cells, whereas SDC-1 silencing only affects wound healing.Malignant mesothelioma (MM) is an aggressive tumor of the serosal cavities. Angiogenesis is important for mesothelioma progression, but so far, anti-angiogenic agents have not improved patient survival. Our hypothesis is that better understanding of the regulation of angiogenesis in this tumor would largely improve the success of such a therapy. Syndecan-1 (SDC-1) is a transmembrane heparan sulfate proteoglycan that acts as a co-receptor in various cellular processes including angiogenesis. In MM, the expression of SDC-1 is generally low but when present, SDC-1 associates to epithelioid differentiation, inhibition of tumor cell migration and favorable prognosis, meanwhile SDC-1 decrease deteriorates the prognosis. In the present study, we studied the effect of SDC-1 overexpression and silencing on MM cells ability to secrete angiogenic factors and monitored the downstream effect of SDC-1 modulation on endothelial cells proliferation, wound healing, and tube formation. This was done by adding conditioned medium from SDC-1 transfected and SDC-1 silenced mesothelioma cells to endothelial cells. Moreover, we investigated the interplay and molecular functional changes in angiogenesis in a co-culture system and characterized the soluble angiogenesis-related factors secreted to the conditioned media. We demonstrated that SDC-1 over-expression inhibited the proliferation, wound healing, and tube formation of endothelial cells. This effect was mediated by a multitude of angiogenic factors comprising angiopoietin-1 (Fold change ± SD: 0.65 ± 0.07), FGF-4 (1.45 ± 0.04), HGF (1.33 ± 0.07), NRG1-β1 (1.35 ± 0.08), TSP-1 (0.8 ± 0.02), TIMP-1 (0.89 ± 0.01) and TGF-β1 (1.35 ± 0.01). SDC-1 silencing increased IL8 (1.33 ± 0.06), promoted wound closure, but did not influence the tube formation of endothelial cells. Pleural effusions from mesothelioma patients showed that Vascular Endothelial Growth Factor (VEGF) levels correlate to soluble SDC-1 levels and have prognostic value. In conclusion, SDC-1 over-expression affects the angiogenic factor secretion of mesothelioma cells and thereby inhibits endothelial cells proliferation, tube formation, and wound healing. VEGF could be used in prognostic evaluation of mesothelioma patients together with SDC-1.

Study of TGFβ‐induced modifications in central and peripheral endothelium of normal corneas

AbstractPurposeTGF‐βs trigger ENdothelial‐Mesenchymal Transition (EnMT) in corneal endothelial cells (ECs) in animal models of endothelial wound healing. All the 3 isoforms of TGF‐β were detected in human aqueous humor and their receptors were detected in ECs. More and more evidence indicated that central and peripheral ECs have a different status.AimTo compare the effects of the 3 isoforms of TGF‐β1, 2 and 3 on ex vivo corneal endothelium and to compare their effects on central and peripheral endothelium.Methods10 pairs of fresh human corneas (body donation to Sciences) were incubated in a serum free medium for 2 weeks at 31°C. One cornea of each pair was incubated with 10 ng/mL of TGF β1 or 2 or 3, the paired cornea without TGF‐β or with another isoform of TGF‐β (n = 2 for each comparison). Immunolabelling using markers of ECs (ZO‐1, CD166, COX IV and N‐cadherin) and of mesenchymal cells (MCs) (CD44, Collagen 1 and fibronectin) was performed of flat‐mounted corneas. Staining was observed with confocal microscopy to map the regional expression of each marker.ResultsIn the controls, MC markers were expressed only by ECs located at the extreme periphery and by trabecular cells. The 3 isoforms of TGF‐β induced the loss of EC markers and the increase of MC markers in central and peripheral endothelium. The effects of TGF‐β1 and 2 were higher than that of TGF‐β3. TGF‐β‐induced alterations were more pronounced in central than in peripheral endothelium.ConclusionsNormal ECs at the extreme periphery and trabecular cells share characteristics of MCs. These ECs could then derive from trabecular cells. TGF‐β (10 ng/ml) induces EnMT in the center of the endothelium of normal corneas. Physiological concentration of TGF‐β might contribute to maintain peripheral ECs in an undifferentiated state able to slowly proliferate and contribute to endothelial homeostasis.

Exosomes secreted from mesenchymal stem cells mediate the regeneration of endothelial cells treated with rapamycin by delivering pro-angiogenic microRNAs

Delayed endothelial healing after drug eluting stent (DES) implantation is a critical clinical problem in treatment of coronary artery diseases. Exosomes exhibit proangiogenic potential in a variety of ischemic diseases. However, the association of exosomes with endothelial regeneration after DES implantation has been rarely reported. In this study, we aimed to investigate the therapeutic effects of mesenchymal stem cell (MSC)-derived exosomes on endothelial cells treated with rapamycin and explore the potential mechanisms of MSC-derived exosomes in promoting endothelial regeneration. Exosomes were isolated from MSCs by ultracentrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot assay. The in vitro effects of MSC-derived exosomes on the proliferation and migration of endothelial cells treated with rapamycin were evaluated by integrated experiment, cell counting kit-8, scratch, tube formation, and transwell assays. And the apoptosis of rapamycin-induced endothelial cells loaded with MSC-derived exosomes was detected using TUNEL and Annexin-V FITC and PI double-staining assays. The microRNA (miRNA) cargo of MSC-derived exosomes was identified by high-throughput RNA sequencing. Pro-angiogenic miRNAs and key pathways were further characterized. Our results indicated that MSC-derived exosomes could be ingested into umbilical vein endothelial cells (HUVECs) and significantly enhanced cell proliferation rate, migratory and tube-forming capabilities in vitro. MSC-derived exosomes also inhibited the apoptosis of HUVECs induced by rapamycin. A distinct class of exosomal miRNAs was further identified, including six miRNAs tightly related to neovasculogenesis. Silencing the expression of exosomal miRNA-21–5p and let-7c-5p attenuated the pro-proliferative and pro-migratory capacity of MSC-derived exosomes. Moreover, functional enrichment analysis indicated that metabolic pathways might contribute to reendothelialization. This study highlights a proregenerative effect of MSC-derived exosomes in vitro, which may be partly explained by the delivery of pro-angiogenic miRNAs to endothelial cells.

Evaluation of corneal endothelial cell changes in diabetic patients before and after phacoemulsification surgery: A comparative study

Aim: The study was conducted to evaluate and compare the changes in corneal endothelial cell count density (ECD) and Central corneal thickness (CCT) between diabetic and non diabetic population before and after phacoemulsification surgery. Settings and Design: This prospective, comparative, interventional study was carried out at government medical college and attached group of hospitals, a tertiary eye care centre in Rajasthan, India. Materials and Methods: A total of 50 patients with type II diabetes mellitus (age group 30-80 years) and 50 age matched healthy patients of senile cataract with clear cornea without any other ocular pathology were non-randomly selected for Phacoemulsification surgery (phaco) after detailed preoperative assessment. All patients were operated by the same surgeon with phaco chop technique to exclude any surgeon’s related bias. Endothelial cell count, CCT and Coefficient of Variance (CV) was measured with specular microscope pre-operatively and postoperatively at day 1st, 7th, 15th, 1 month, and 3 months serially. Result: Postoperatively, the mean ECD was decreased in both the groups but loss of endothelial cell count was significantly more in diabetic population as compare to non diabetic groups at day 1, day 7, day 15, 1 month and 3 months. CCT was increased on first post-operative day in both the groups, subsequently decreased and then returned back to about same as pre-operative state after 1 month. But postoperatively, the change in mean CCT was not significant between both groups. The difference in % CV was significantly higher in diabetic group as compared to non diabetic group postoperatively. Conclusion: The corneal endothelial cell changes were significantly high in diabetic population as compared to nondiabetic patients. The ECD loss, increase in mean CCT and % CV were more in diabetic population as compared to non diabetic. And patients with diabetes showed slower recovery in endothelial healing as compared to non diabet

In This Issue

HomeCirculation ResearchVol. 127, No. 12In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published3 Dec 2020https://doi.org/10.1161/RES.0000000000000452Circulation Research. 2020;127:1469is related toArterial Stiffness Preceding DiabetesTamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle CellsATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and FunctionTamoxifen and E2 Effects on Reendothelialization (p 1473)Zahreddine et al investigate Tamoxifen’s vasculoprotective effects.Download figureDownload PowerPointFor breast cancers that contain high levels of estrogen receptor, a standard treatment is to give drugs that block either estrogen production, or the receptor itself, such as Tamoxifen. Eliminating the vasculoprotective effects of estrogen might increase the risk of cardiovascular disease. But because Tamoxifen can antagonize or activate the receptor depending on the tissue, its role in such risk is unclear. Some evidence even suggests it might have protective effects, such as promoting vascular endothelial healing. Zahreddine and colleagues now show that while mice suffering endovascular injury—damage to the inside of a blood vessel—have improved healing when treated with Tamoxifen or with estrogen, those suffering pervascular injury—which also affects the surrounding smooth muscle—heal only in response to estrogen. This suggested Tamoxifen’s healing effect might require smooth muscle cells (SMCs). Sure enough, mice lacking the estrogen receptor in SMCs, healed endovascular injuries in response to the hormone but not Tamoxifen, while mice lacking the receptor in endothelial cells, healed such injuries in response to Tamoxifen but not estrogen. The work reveals nuance in the molecular actions of Tamoxifen that should inform future assessments of its risks and benefits, say the authors.Arterial Stiffness Preceding Diabetes (p 1491)Having stiffer arteries may increase a person’s risk for diabetes, say Zheng et al.Download figureDownload PowerPointAs a person ages, their risk of developing diabetes and cardiovascular disease increases. Aging is also linked to increasing arterial stiffness and high blood pressure, but how all these individual conditions affect and influence each other is not entirely clear. For example, while arterial stiffness and diabetes tend to correlate, whether one increases the risk for the other, or the risk relationship is bidirectional, is not known. To find out, Zheng and colleagues studied diabetes and arterial stiffness in a cohort of 8,956 Chinese people between 2010 and 2015, none of whom had diabetes or cardiovascular disease at the outset. With repeated measures of fasting glucose levels—as an indicator of diabetes—and pulse wave velocity—as a measure of arterial stiffness—the team found that participants with a higher baseline arterial stiffness were more likely to develop diabetes during the five-year period than those with lower stiffness. A total of 979 individuals developed diabetes during the study. Higher baseline glucose levels did not predict future arterial stiffness, however, suggesting the risk relationship is one-way. While the results require confirmation in further cohorts, they also set the stage for investigations into pathological mechanisms linking arterial stiffness to diabetes.ATAC-seq Identifies Novel Isl1 SAN Enhancer (p 1502)Galang et al discover a novel sinoatrial node enhancer element.Download figureDownload PowerPointPacemaker cells (PCs) of the sinoatrial node establish and control the rhythmic contractions of the heart. These cells differ from regular cardiomyocytes in their transcription profiles, but how this transcriptional fate is established and maintained is not fully understood. To investigate the epigenetic landscape defining PC fate, Galang and colleagues have employed a technique called ATAC-seq, which identifies areas of the genome with accessible, open chromatin structures—an indication of transcriptional activity. The team compared the genomes of PCs and atrial cardiomyocytes and found a number of PC-specific accessible loci that had both large numbers of transcription factor binding sequences and enhancer activity when assayed in mice. The team went on to specifically characterize one novel enhancer upstream of the gene encoding Isl1—a key transcription factor for PC identity. They showed that deleting the enhancer caused underdevelopment of the sinoatrial node and arrhythmias in mice. They also noted that single nucleotide polymorphisms at the equivalent locus in humans had been linked to variations in resting heart rate. The report verifies ATAC-seq as an effective tool for identifying PC enhancers, and will launch further studies into how such enhancers function in heart development and disease. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesArterial Stiffness Preceding DiabetesMengyi Zheng, et al. Circulation Research. 2020;127:1491-1498Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle CellsRana Zahreddine, et al. Circulation Research. 2020;127:1473-1487ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and FunctionGiselle Galang, et al. Circulation Research. 2020;127:1502-1518 December 4, 2020Vol 127, Issue 12Article InformationMetrics Download: 308 © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000452 Originally publishedDecember 3, 2020 PDF download

Effects of IL-1β and TNF-α on the Expression of P311 in Vascular Endothelial Cells and Wound Healing in Mice.

ObjectiveThis study aimed to define the role of interleukine-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the expression of P311 in vascular endothelial cells (VECs) and in wound healing.MethodsDAPI staining, a CCK-8 assay, cell migration assay, and an angiogenesis assay were used to assess the effects exerted by TNF-α and IL-1β at various concentrations on morphology, proliferation, migration, and angiogenesis of VECs. Western blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) models were employed to observe the effects exerted by proteins related to the nuclear factor-kappa B (NF-κB) signaling pathway and P311 mRNA expression. Bioinformatics analysis was performed on the binding sites of P311 and NF-κB. Finally, to investigate the effects of IL-1β and TNF-α on wound healing and the length of new epithelium in mice, we established a full-thickness wound defect model in mice. Immunohistochemistry was used to measure changes in P311, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), as well as other related proteins.ResultsWhen levels of TNF-α and IL-1β were both 20 ng/ml, their effects on cell proliferation, cytoskeleton protein expression, cell migration, and angiogenesis were the greatest (P < 0.05). IL-1β and TNF-α at moderate concentrations effectively promoted P311 mRNA and p-NF-κB protein expression (P < 0.05), while p-NF-K b protein expression was decreased (P < 0.05). Luciferase assays showed that P311 expression was also relatively greater when stimulated at moderate concentrations (P < 0.05), while relative expression was significantly lower when the p-NF-K b inhibitor CAPE was added (P < 0.05). On 7-day wound healing rate comparison, the control, IL-1β, IL-1βab, TNF-α, and TNF-αab groups were 18, 37, 35, 39, and 36%, respectively, while control group + P311 siRNA was 31% (P < 0.05). New epithelial length, granulation tissue thickness, and number of blood vessels trends were also the same. In the control group, P311 showed lower relative expression levels than the others (P < 0.05). P311 relative expression levels trended as follows: control group > IL-1βab > IL-1β > TNF-αab > TNF-α (P < 0.05).ConclusionWhen IL-1β and TNF-α concentrations are moderate, they effectively promote the proliferation, expression, migration, and angiogenesis of VECs, possibly by promoting the expression of the NF-K b pathway and thereby promoting the expression of P311. In vitro experiments on mice suggest that P311 effectively promotes wound healing, and its mechanism may be closely related to PCNA, CD31, and VEGF.

Thromboresistance and endothelial healing in polymer-coated versus polymer-free drug-eluting stents: Implications for short-term dual anti-platelet therapy

BackgroundShort-term dual antiplatelet therapy (DAPT) is a suitable strategy after stent implantation especially in patients at high risk for bleeding. The thromboresistant characteristics and the healing profile permanent polymer stents such as the Resolute Onyx- drug-eluting stent (DES) has never been tested against the current approved stents for short-term DAPT, the polymer free (PF) biolimus-eluting stent (PF-BES) and bare metal stents (BMS) in dedicated preclinical models. MethodsAn ex-vivo porcine arteriovenous shunt and in-vivo flow loop model were used to evaluate thromboresistance. The healing profile was assessed in the rabbit model at 28 days by confocal microscopy (CM), scanning electron microscopy (SEM) and histology. Onyx-DES was separately compared with Onyx-BMS in first experiment and PF-BES in second experiment. ResultsIn an ex-vivo shunt model, CM and SEM showed significantly less platelet adhesion for Onyx-DES relative to Onyx-BMS and PF-BES. In a flow loop model using human blood, platelet adhesion was also significantly less in Onyx-DES as compared to PF-BES and Onyx-BMS. In the healing study, Onyx-BMS showed significantly greater healing profile relative to Onyx-DES as expected, whereas Onyx-DES showed equivalent endothelial coverage by SEM and significantly less Evan's blue uptake and comparable colocalization of p120 and vascular endothelial-cadherin when compared with PF-BES. ConclusionsOnyx-DES showed qualities of thomboresistance and healing which appear to be compatible with short-term DAPT. Thromboresistance was superior to PF-BES and healing was equivalent to PF-BES in this pre-clinical study. Onyx-DES might provide advantages when considering short-term DAPT especially in patients at high risk of bleeding.

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Endothelial response boosted by platelet lysate: The involvement of calcium toolkit

Wound repair is a dynamic process during which crucial signaling pathways are regulated by growth factors and cytokines released by several kinds of cells directly involved in the healing process. However, the limited applications and heterogeneous clinical results of single growth factors in wound healing encouraged the use of a mixture of bioactive molecules such as platelet derivatives for best results in wound repair. An interesting platelet derivative, obtained from blood samples, is platelet lysate (PL), which has shown potential clinical application. PL is obtained from freezing and thawing of platelet-enriched blood samples. Intracellular calcium (Ca2+) signals play a central role in the control of endothelial cell survival, proliferation, motility, and differentiation. We investigated the role of Ca2+ signaling in the PL-driven endothelial healing process. In our experiments, the functional significance of Ca2+ signaling machinery was highlighted performing the scratch wound assay in presence of different inhibitors or specific RNAi. We also pointed out that the PL-induced generation of intracellular ROS (reactive oxygen species) via NOX4 (NADPH oxidase 4) is necessary for the activation of TRPM2 and the resulting Ca2+ entry from the extracellular space. This is the first report of the mechanism of wound repair in an endothelial cell model boosted by the PL-induced regulation of [Ca2+]i.

HMGA2-mediated tumorigenesis through angiogenesis in leiomyoma

To study the role of HMGA2 in promoting angiogenesis in uterine leiomyoma (LM). This study involved evaluation of vessel density and angiogenic factors in leiomyomas with HMGA2 overexpression; examining angiogenic factor expression and AKT signaling in myometrial (MM) and leiomyoma cells by introducing HMGA2 overexpression invitro; and exploring vessel formation induced by HMGA2 overexpression both invitro and invivo. University research laboratory. None. None. The main outcome measures include vessel density in leiomyomas with HMGA2 (HMGA2-LM) or MED12 (MED12-LM) alteration; angiogenic factor expression in primary leiomyoma and invitro cell line model; and vessel formation in leiomyoma cells with HMGA2 overexpression invitro and invivo. Angiogenic factors and receptors were significantly upregulated at mRNA and protein levels in HMGA2-LM. Specifically, HMGA2-LM exhibited increased expression of VEGFA, EGF, bFGF, TGFα, VEGFR1, and VEGFR2 compared to MED12-LM and myometrium. Overexpression of HMGA2 in MM and LM cell lines resulted in increased secretion of angiogenesis-associated factors. Secreted factors promoted human umbilical vein endothelial cell (HUVEC) migration, tube formation, and wound healing. HMGA2 overexpression upregulated IGF2BP2 and pAKT, and silencing the IGF2BP2 gene reduced pAKT levels and reduced HUVEC migration. Myometrial cells with stable HMGA2 overexpression exhibited increased colony formation and cell growth invitro and formed xenografts with increased blood vessels. HMGA2-LM have a high vasculature density, which likely contributes to tumor growth and disease burden of this leiomyoma subtype. HMGA2 plays an important role in angiogenesis and the involvement of IGF2BP2-mediated pAKT activity in angiogenesis, which provides a potential novel target for therapy for this subtype of LM.

Comparison of Endothelial Barrier Functional Recovery After Implantation of a Novel Biodegradable-Polymer Sirolimus-Eluting Stent in Comparison to Durable- and Biodegradable-Polymer Everolimus-Eluting Stents

AimsThe advantage of biodegradable-polymer drug-eluting stents (BP-DES) versus durable-polymer (DP) DES remains uncertain. We compared neointimal formation and endothelial barrier function of new BP sirolimus-eluting stents (BP-SES, BuMA Supreme®) to other contemporary BP-DES, DP-DES, and bare metal stents (BMS). Methods and resultsLight microscopic assessment in swine coronary arteries showed comparable neointimal formation between BP-SES and DP everolimus-eluting stent (DP-EES). The performance of BP-SES was compared with DP-EES (Xience Xpedition®), BP-EES (Synergy®), and BMS (Multi-Link Vision®) at 45- and 90-days in rabbit ilio-femoral arteries using Evans blue dye (EBD) followed by immunostaining for endothelial barrier proteins (p120/vascular endothelial-cadherin [VE-cad]) to evaluate endothelial barrier function and scanning electron microscopy (SEM) to determine strut tissue coverage. BMS followed by BP-SES and BP-EES exhibited smaller EBD positive areas versus that of DP-EES at 45- and 90-days. p120/VE-cad immunostaining and SEM-determined strut coverage was greater at 45- and 90-days for BMS followed by all DESs. Regardless of stent type, the lack of p120/VE-cad co-localization showed greater leukocyte and platelet aggregation. ConclusionThree types of DES showed different endothelial healing pattern regardless their equivalent suppression of neointimal formation.

Abstract 413: Inhibition of Phospholipase A2 Preserves Endothelial Cell Migration in Presence of Lipid Oxidation Products

During vascular intervention oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, which inhibits endothelial cell (EC) migration, and impedes endothelium healing. We have previously shown that lysoPC activates canonical transient receptor potential 6 (TRPC6) channel that leads to a prolonged [Ca2+]i influx, causing the inhibition in EC migration. We hypothesize that lysoPC activates phospholipase A 2 (PLA 2 ) crucial for TRPC6 activation; PLA 2 acts on cellular membranes to release arachidonic acid that opens arachidonate regulated calcium channel to release the initial calcium required to trigger TRPC6, blocking PLA 2 will prevent the opening of TRPC6 channels, preserve migration and promote endothelium healing. After incubation of bovine aortic EC with ATK or Ax048 to block PLA2G4, or with BEL, FKGK11, or FKGK18 to block PLA2G6, the effect of lysoPC-induced TRPC6 externalization and EC migration was assessed. Reversible PLA2G6 pharmacological inhibitors maximally blocked lysoPC-induced TRPC6 externalization, arachidonic acid release and preserved EC migration; exemplified with biotinylation assay, arachidonate enzyme assay, and razor scrape migration assay respectively. Immunofluorescence microscopy for TRPC6 plasma membrane translocation and PLA 2 enzyme activity assay supported these findings. To further verify the specific isoforms involved in blocking TRPC6 externalization, siRNA mediated transient knockdown studies were performed with PLA2G4A, PLA2G4C, PLA2G6A, and PLA2G6B siRNAs in EA.hy926— human endothelial cell line. PLA2G6A and not PLAG6B/4A/4C downregulation completely blocked TRPC6 externalization and preserved migration comparable to control levels. These studies confirmed the role of PLA2G6A (a calcium-independent cytosolic phospholipase A 2 group VI β isoform) in blocking lysoPC-induced TRPC6 activation and preservation of EC migration. Additional studies are in progress to confirm in vivo relevance of these findings. Our results show the potential for developing PLA 2 targeted therapies to block TRPC6 activation and promote endothelium healing, thus improving the outcomes for patients undergoing cardiovascular intervention.