Primary Cultures Of Endothelial Cells Research Articles

CD133+CD34+ cells can give rise to EPCs: A comparative rabbit and human study

• "Blood-island" like cell clusters is not the only pattern in the emergence of EPCs from MNCs during culturing. • We identified two more distinct attributes, biphasic EPCs and de novo EPCs. • EPCs might coexist as a subset in the MNCs. • CD34 + CD133 + cells from both rabbit and human primary endothelial cell culture can give rise to EPCs. • CD133 is subsequently lost during progression as a cell line, confirming that it is an early cell marker.

Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pHi

Recent studies have shown that lactate coupled water flux is the underlying mechanism of the corneal endothelial pump, which is highly dependent on the presence of bicarbonate. In this study we test the hypothesis that the increased intracellular pH (pHi) caused by bicarbonate stimulates glycolytic activity and the production of lactate by endothelial cells. Primary cultures of bovine corneal endothelial cells (BCEC) were incubated in bicarbonate-free (BF) ringer, a high [HEPES] ringer, and bicarbonate-rich (BR) ringer all at pH 7.5. Lactate production and glucose consumption were greatest in BR>HEPES >BF. Similarly, pHi was greatest in BR>HEPES>BF. Increasing pHi with NH4Cl also increased lactate production in BF or BR, indicating that the increased lactate production in BR is not due to HCO3− itself. Glucose transport capacity, as measured by 2-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino-2-Deoxyglucose (2-NBDG) uptake was unaffected by the three incubation conditions. Using Laconic, a FRET sensor for lactate, we found that intracellular [lactate] increased immediately and transiently when cells were switched from BF to BR perfusion indicating increased lactate production with subsequent matching of efflux. Moreover, induction of acute lactate influx by perfusion pulses of 10 mM lactate increased intracellular [lactate] significantly faster in BF than in BR, consistent with higher lactate production and efflux in BR. In summary, our results indicate that glycolytic flux and lactate production increase in BR due to increased pHi, consistent with the well-known pH sensitivity of phosphofructokinase, the rate limiting enzyme in glycolysis.

Clinopodium tomentosum (Kunth) Govaerts Leaf Extract Influences in vitro Cell Proliferation and Angiogenesis on Primary Cultures of Porcine Aortic Endothelial Cells.

Clinopodium tomentosum (Kunth) Govaerts is an endemic species in Ecuador, where it is used as an anti-inflammatory plant to treat respiratory and digestive affections. In this work, effects of a Clinopodium tomentosum ethanolic extract (CTEE), prepared from aerial parts of the plant, were investigated on vascular endothelium functions. In particularly, angiogenesis activity was evaluated, using primary cultures of porcine aortic endothelial cells (pAECs). Cells were cultured for 24 h in the presence of CTEE different concentrations (10, 25, 50, and 100 μg/ml); no viability alterations were found in the 10-50 μg/ml range, while a slight, but significant, proliferative effect was observed at the highest dose. In addition, treatment with CTEE was able to rescue LPS-induced injury in terms of cell viability. The CTEE ability to affect angiogenesis was evaluated by scratch test analysis and by an in vitro capillary-like network assay. Treatment with 25-50 μg/ml of extract caused a significant increase in pAEC's migration and tube formation capabilities compared to untreated cells, as results from the increased master junctions' number. On the other hand, CTEE at 100 μg/ml did not induce the same effects. Quantitative PCR data demonstrated that FLK-1 mRNA expression significantly increased at a CTEE dose of 25 μg/ml. The CTEE phytochemical composition was assessed through HPLC-DAD; rosmarinic acid among phenolic acids and hesperidin among flavonoids were found as major phenolic components. Total phenolic content and total flavonoid content assays showed that flavonoids are the most abundant class of polyphenols. The CTEE antioxidant activity was also showed by means of the DPPH and ORAC assays. Results indicate that CTEE possesses an angiogenic capacity in a dose-dependent manner; this represents an initial step in elucidating the mechanism of the therapeutic use of the plant.

Abstract 5240: Effective inhibition of TMPRSS2-ERG positive prostate cancer cells by a new formula of ERGi-USU-6

Abstract Background: Prostatic adenocarcinoma is among the leading cause of cancer-related deaths among men in the United States. The Erythroblast Transformation-Specific-Related Gene, ERG is dormant in normal prostate epithelium. Due to gene fusions, the ERG oncogene is frequently activated by androgenic signals in prostate cancer. ERG disrupts the normal differentiation, promotes epithelial-mesenchymal transition, migratory and invasive properties of cancer cells. Approximately 35% of metastatic castration-resistant prostate cancers harbor ERG oncogene. Due to the failure of androgen axis directed therapies, there is an urgent need to develop inhibitors to targeting prostate cancer-causing genes, such as ERG. We have identified a potent small-molecule inhibitor ERGi-USU that is remarkably selective for inhibiting the growth of ERG positive cancer cells through direct binding to the RIOK2 atypical kinase, a putative upstream regulator of ERG. We completed a structure-activity relationship (SAR) study and compound development resulting in the potent derivative, ERGi-USU-6. Our current objective is to improve the therapeutic properties of ERGi-USU-6, by new salt formulations. Methods: Evaluation of the five selected salt formulations of ERGi-USU-6 were performed by assessing the growth of ERG positive prostate cancer cell line (VCaP) and by monitoring ERG and RIOK2 protein levels. Selectivity was assessed by monitoring the growth, endogenous ERG and RIOK2 levels in normal ERG positive human umbilical vein derived endothelial cells (HUVEC). The IC50 values for cell growth, compared to the parental compound were calculated in a 12-step dilution range performed in triplicates and independently repeated three times. Cell growth was measured by quantitative Cell Glow assay monitoring viable cells using Perkin Elmer Envision assay instrument and protein levels were quantified by measuring bioluminescence in IBright instrument in a wide dynamic range. Results: One new salt formula with improved activity were identified, demonstrating improved cell growth (IC50=89nM vs. parental IC50=139), ERG protein and RIOK2 protein inhibition. None of the salt formulas of ERGi-USU-6 showed any effect on the primary cultures of the ERG positive normal endothelial cells (HUVEC) in the effective concentration range. Conclusion: The first evaluation of salt formulas of ERGi-USU-6 may open the possibilities for preclinical assessments of this remarkably cancer-selective compound. Citation Format: Binil Eldhose, Charles P. Xavier, Mallesh Pandrala, Sanjay V. Malhotra, Albert Dobi. Effective inhibition of TMPRSS2-ERG positive prostate cancer cells by a new formula of ERGi-USU-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5240.

Цитотоксичность трусопта и дорзолана соло на первичной культуре эндотелиальных клеток роговицы человека

Изучено влияние антиглаукомных препаратов трусопт и дорзолан соло, содержащих дорзоламид в качестве действующего вещества в одинаковой концентрации, на культуру эндотелиальных клеток роговицы человека. Выявлено, что трусопт обладает более выраженным цитотоксическим эффектом (снижение выживаемости клеток на 8,5 % при концентрации в нем дорзоламида 20 мкг/мл, (p = 0,022) и на 12,8 % при концентрации в нем дорзоламида 200 мкг/мл (p < 0,0001), по сравнению с препаратом дорзолан соло, что, возможно, обусловлено наличием в его составе бензалкония хлорида.

Acute antioxidant and cytoprotective effects of sulforaphane in brain endothelial cells and astrocytes during inflammation and excitotoxicity.

Sulforaphane (SFN), a bioactive phytochemical isothiocyanate, has a wide spectrum of cytoprotective effects that involve induction of antioxidant genes. Nongenomic antioxidant effects of SFN have not been investigated. Brain oxidative stress during inflammation and excitotoxicity leads to neurovascular injury. We tested the hypothesis that SNF exhibits acute antioxidant effects and prevents neurovascular injury during oxidative stress. In primary cultures of cerebral microvascular endothelial cells (CMVEC) and cortical astrocytes from the newborn pig brain, a pro‐inflammatory cytokine TNF‐α and an excitotoxic glutamate elevate reactive oxygen species (ROS) and cause cell death by apoptosis. Nox4 NADPH oxidase is the main Nox isoform in CMVEC and cortical astrocytes that is acutely activated by TNF‐α and glutamate leading to ROS‐mediated cell death by apoptosis. The Nox4 inhibitor GKT137831 blocked NADPH oxidase activity and overall ROS elevation, and prevented apoptosis of CMVEC and astrocytes exposed to TNF‐α and glutamate, supporting the leading role of Nox4 in the neurovascular injury. Synthetic SFN (10−11‐10−6 mol/L) inhibited NADPH oxidase activity and reduced overall ROS production in CMVEC and astrocytes within 1‐hour exposure to TNF‐α and glutamate. Furthermore, in the presence of SFN, the ability of TNF‐α and glutamate to produce apoptosis in CMVEC and cortical astrocytes was completely prevented. Overall, SFN at low concentrations exhibits antioxidant and antiapoptotic effects in cerebral endothelial cells and cortical astrocytes via a via a nongenomic mechanism that involves inhibition of Nox4 NADPH oxidase activity. SFN may prevent cerebrovascular injury during brain oxidative stress caused by inflammation and glutamate excitotoxicity.

Bioengineering of Human Corneal Endothelial Cells from Single- to Four-Dimensional Cultures

To summarize the recent advances of clinical and preclinical studies for corneal endothelial tissue bioengineering. The challenges facing the generation of a clinical applicable corneal endothelial graft can be broadly classified into cell source selection, culture medium optimization, scaffold establishment, and the following three- and four-dimensional (4D) corneal construction. Based on the current advances in primary human corneal endothelial cell (HCEC) culture and good manufacturing practice (GMP)–compliant medium development, the first clinical trial of bioengineered HCEC injection therapy has been conducted with encouraging results. Other significant findings include the in vivo experiments of the stem cell–derived HCEC, the development of serum-, xeno-, and additive-free media, and the construction of 4D scaffold. It can be anticipated that tissue engineering–based therapy toward corneal endothelial diseases will replace the current keratoplasty method as a promising treatment option in the near future.

All-trans-Retinaldehyde Contributes to Retinal Vascular Permeability in Ischemia Reperfusion.

PurposeExtracellular accumulation of all-trans-retinaldehyde (atRAL), a highly reactive visual cycle intermediate, is toxic to cells of the outer retina and contributes to retinal and macular degenerations. However, the contribution of atRAL to retinal capillary function has not been studied. We hypothesized that atRAL released from the outer retina can contribute to retinal vascular permeability. We, therefore, tested the contribution of atRAL to retinal ischemia-reperfusion (IR)-induced vascular permeability.MethodsIR was induced in mice by transient increase in intraocular pressure followed by natural reperfusion. The visual cycle was ablated in the Lrat−/− mice, reduced by dark adaptation or the use of the RPE65 inhibitor and atRAL scavenger emixustat. Accumulation of FITC-BSA was used to assess vascular permeability and DNA fragmentation quantified cell death after IR. Primary bovine retinal endothelial cell (BREC) culture was used to measure the direct effects of atRAL on endothelial permeability and cell death.ResultsInhibition of the visual cycle by Lrat−/−, dark adaptation, or with emixustat, all reduced approximately half of IR induced vascular permeability at 48 hours. An increase in BREC permeability with atRAL coincided with lactate dehydrogenase (LDH) release, a measure of cell death. Both permeability and toxicity were blocked by emixustat.ConclusionsOuter retinal pathology may contribute to vascular permeability by release of atRAL, which can act directly on vascular endothelial cells to alter barrier properties and induce cell death. These studies may have implications for a variety of blinding eye diseases that include outer retinal damage and retinal vascular permeability.

Inhibitory effect of paeonol on aortic endothelial inflammation in atherosclerotic rats by up-regulation of caveolin-1 expression and suppression of NF-κB pathway

To explore whether paeonol can play an anti-atherosclerotic role by regulating the expression of aortic caveolin-1 and affecting NF-κB pathway, so as to inhibit the inflammatory response of vascular endothelium in atherosclerotic rats. The atherosclerotic model of rats was induced by high-fat diet and vitamin D_2. The primary culture of vascular endothelial cells(VECs) was carried out by tissue block pre-digestion and adherent method. The injury model of VECs was induced by lipopolysaccharide(LPS), and filipin, a small concave protein inhibitor, was added for control. HE staining was used to observe pathological changes of aorta. TNF-α, IL-6 and VCAM-1 were detected by ELISA. Western blot assay was used to detect the protein expression levels of caveolin-1 and p65 in aorta and VECs. The results showed that as compared with model group, paeonol significantly reduced aortic plaque area and lesion degree in rats, decreased the level of serum TNF-α, IL-6 and VCAM-1 in the rats and enhanced the relative expression level of caveolin-1, decreased p65 expression conversely(P<0.05 or P<0.01). In vitro, as compared to model group, paeonol obviously improved cell morphology, decreased the secretion of TNF-α, IL-6 and VCAM-1 in VECs, increased caveolin-1 expression, and decreased p65 protein expression(P<0.05 or P<0.01). Furthermore, filipin could reverse the effect of paeonol on expression of inflammatory factors and proteins(P<0.05 or P<0.01). According to the results, it was found that paeonol could play the role of anti-atherosclerosis by up-regulating the expression of caveolin-1 and inhibiting the activation of NF-κB pathway to reduce vascular inflammation in atherosclerotic rats.

Culture-induced changes in mRNA expression levels of efflux and SLC-transporters in brain endothelial cells

BackgroundThe complexity of the neurovascular unit (NVU) poses a challenge in the investigations of drug transport across the blood–brain barrier (BBB) and the function of the brain capillary endothelium. Several in vitro models of the brain capillary endothelium have been developed. In vitro culture of primary endothelial cells has, however, been reported to alter the expression levels of various brain endothelial proteins. Only a limited number of studies have addressed this in detail. The aim of the present study was to investigate mRNA levels of selected BBB transporters and markers in in vitro models of the BBB based on bovine primary endothelial cells and compare these to the levels estimated in freshly isolated bovine brain capillaries.MethodsBrain capillaries were isolated from bovine cerebral cortex grey matter. Capillaries were seeded in culture flasks and endothelial cells were obtained using a brief trypsinization. They were seeded onto permeable supports and cultured in mono-, non-contact- or contact co-culture with/without primary rat astrocytes. mRNA-expression levels of the selected BBB markers and transporters were evaluated using qPCR and monolayer integrity of resulting monolayers was evaluated by measuring the transendothelial electrical resistance (TEER).ResultsThe capillary mRNA transcript profile indicated low expression of ABCC1 and CLDN1. The mRNA expression levels of TPA, OCLN, ABCB1, SLC2A1, SLC16A1 and SLC7A5 were significantly decreased in all culture configurations compared to freshly isolated bovine brain capillaries. ALP, VWF, ABCC1 and ABCC4 were upregulated during culture, while the mRNA expression levels of F11R, TJP1, CLDN5, CLDN1 and ABCG2 were found to be unaltered. The mRNA expression levels of VWF, ALP, ABCB1 and ABCC1 were affected by the presence of rat astrocytes.ConclusionThe endothelial mRNA transcript profile in bovine capillaries obtained in this study correlated nicely with profiles reported in mice and humans. Cultured endothelial cells drastically downregulated the mRNA expression of the investigated SLC transporters but maintained expression of efflux transporter and junctional protein mRNA, implying that the bovine in vitro BBB models may serve well to investigate basic barrier biology and in vivo permeation of passively permeating compounds and efflux transporter substrates but may be less well suited for investigations of SLC-mediated transport.

Control of Vasoconstriction by Myoendothelial Ca2+ Signaling Through Gap Junctions

In resistance arteries, smooth muscle cells (SMCs) and endothelial cells (ECs) are communicated through gap junctions (i.e. myoendothelial gap junctions), which provide a signaling pathway for control of vasomotor tone. In this context, the Ca2+ signaling associated to smooth muscle contraction might also be transmitted via myoendothelial gap junctions and activate a regulatory feedback mechanism (myoendothelial feedback) by the production of endothelium‐dependent vasodilators. It is thought that IP3 is the main Ca2+‐related signaling transmitted from smooth muscle cells to endothelial cells, but the direct contribution of Ca2+ to the myoendothelial feedback is not clear. We investigated the temporal pattern of vasoconstriction in mesenteric resistance arteries, and their temporal relationship with the increase of Ca2+ in SMC and EC. We used intact mesenteric resistance arteries of Sprague dawley male rats (≤ 10 mm in length) to measure the vasoconstriction and changes in [Ca2+]i of smooth muscle cells and endothelial cells observed in response to phenylephrine (PE) or KCl using the fluorescent indicators of Ca2+, Fluo‐4 and X‐Rhod‐1.Changes in [Ca2+]i were also evaluated in primary cultures of mesenteric endothelial cells. All procedures were approved by the local Bioethical Committee (ID:180806005). The vasoconstriction induced by PE and KCl was not only associated with an increase of [Ca2+]i in smooth muscle cells, but also in endothelial cells, which was prevented by the blockade of L‐type voltage‐dependent Ca2+ channels with 10 μM nifedipine or the inhibition of gap junctions with 50 μM 18‐b‐Glycyrrhetenic acid. Although the stimulation with PE or KCl did not evoke an increment of [Ca2+]i in primary cultures of endothelial cells, the response induced by these vasoconstrictors was larger after blocking nitric oxide (NO) production with NG‐nitro‐L‐arginine (100 μM). These results suggest that the direct diffusion of Ca2+ contributes to the NO‐mediated component of the myoendothelial feedback.Support or Funding InformationConicyt 21171840

Norrin restores blood-retinal barrier properties after vascular endothelial growth factor–induced permeability

Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through β-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/β (GSK-3α/β) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a β-catenin-dependent manner.

Restoration of the Blood‐Spinal Cord Barrier after Spinal Cord Injury through 5‐HT1F Receptor Mediated Mitochondrial Biogenesis

Vascular and mitochondrial dysfunction are well‐established consequences of spinal cord injury (SCI). Evidence suggests mitigating these dysfunctions may be an effective approach in treating SCI. We propose to restore vascular and mitochondrial dysfunction following SCI through pharmacological induction of mitochondrial biogenesis (MB). We previously reported that 5‐hydroxytryptamine receptor 1F (5‐HT1F) agonism induces MB in the spinal cord and promotes locomotor recovery with drug initiation beginning at a clinically relevant time point of 8h post‐SCI. The goal of this study was to elucidate if MB induction with the newer selective 5‐HT1F receptor agonist lasmiditan can enhance restoration of the blood‐spinal cord barrier (BSCB) after SCI. C57BL/6J mice were subjected to moderate SCI using a force‐controlled impactor‐induced contusion model followed by daily administration of lasmiditan (0.1 mg/kg, i.p.) beginning 1h after injury. Integrity of the BSCB was assessed using Evans Blue dye extravasation. SCI increased dye extravasation at 1, 3, and 7d, and lasmiditan‐treated mice displayed reduced dye accumulation at 7d post‐SCI, suggesting accelerated BSCB recovery. Levels of tight junction proteins occludin and zonula occludens‐1 were decreased in vehicle‐treated mice and returned to sham levels with lasmiditan treatment by d7. These findings led us to investigate if lasmiditan induces MB in endothelial cells. Primary cultures of cerebral microvascular endothelial cells from C57BL6 mice were treated with lasmiditan. A Seahorse Biosciences flux analyzer was used to quantify FCCP‐uncoupled oxygen consumption rate (OCR), a marker of MB. Lasmiditan increased FCCP‐uncoupled OCR, nuclear‐ and mitochondrial‐encoded proteins (PGC‐1a, COX1, TFAM), and mitochondrial number, confirming MB. Lastly, treatment with lasmiditan enhanced endothelial branching morphogenesis and migration, indicating a role of the 5‐HT1F receptor activation in angiogenic pathways. Collectively, these data reveal that 5‐HT1F receptor agonist‐induced MB occurs in endothelial cells and may accelerate restoration of the BSCB after SCI.Support or Funding InformationThis study was supported by the National Institutes of Health National Institute of General Medical Sciences: GM084147 (R.G.S), and the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs: BX: 000851 (R.G.S.).

Polarized Intracellular Endothelial Pannexin 3 Can Regulate Vascular Function

Pannexin 3 (Panx3) expression is generally thought to be restricted to skin, cartilage and bone where it is routinely observed on intracellular compartments. However, multiple RNAseq studies have demonstrated consistent expression of Panx3 in endothelium throughout the vasculature. A physiological role for endothelial Panx3 has yet to be described. To assess the expression of Panx3 in intact endothelium, we first performed immunogold labelling on high pressure frozen third‐order mesenteric arteries. Panx3 preferentially localized to internal membranes in transverse sections of the endothelium. Next, we used multiple Panx3 antibodies raised against different topographical locations on third‐order mesenteric arteries prepared en face. We found that endothelial Panx3 was polarized downstream of blood flow, co‐localizing with a subset of calnexin‐positive perinuclear membranes, indicating association with the endoplasmic reticulum (ER). To test whether Panx3 was flow‐sensitive, primary cultures of human endothelial cells were seeded in parallel plate flow chambers and exposed to laminar shear stress (10 dynes/cm2 for 18 hours). Panx3 was polarized in the direction of flow similar to that seen in intact resistance arteries. To interrogate the physiological role of endothelial Panx3, we generated an inducible, cell‐specific Panx3 knockout mouse (Panx3fl/fl/Cdh5‐CreERT2+), which reduces both Panx3 mRNA and protein following tamoxifen injection. Panx3fl/fl/Cdh5‐CreERT2+ mice had normal blood cell parameters, heart function, body length, body weight, and fat content. Because we identified Panx3 in the ER perinuclear region, we began by assessing intracellular calcium on intact third‐order mesenteric arteries. Loss of endothelial Panx3 reduced intracellular calcium activity in endothelium under unstimulated conditions. Because calcium is vital to arterial dilation, we performed pressure myography. We found that resistance arteries from Panx3fl/fl/Cdh5‐CreERT2+ mice exhibit significantly enhanced vasoconstriction in response to phenylephrine, indicating impaired endothelial negative feedback on the smooth muscle cells. Due to the role of resistance arteries in blood pressure regulation, we hypothesized there may be a change in the blood pressure of these mice. We used radiotelemetry and found Panx3fl/fl/Cdh5‐CreERT2+ mice were hypertensive compared to tamoxifen‐injected littermate controls. The sum of this data suggests that endothelial Panx3 is polarized on the ER where it can regulate calcium activity in the endothelium, independent of ATP release, and may be important for vascular function.Support or Funding InformationHL149221 (AGW) and HL120840 (BEI)

Phenotypic and Functional Characterization of human Umbilical Vein Endothelial Cells (hUVEC) Cultures Established under Physiologic vs. Atmospheric Oxygen Tension

Primary cultures of human umbilical vein endothelial cells (hUVEC) have historically been established at atmospheric oxygen (O2) tension (21%) and their surface marker profiles and angiogenic potential in are understood. However, physiologic O2 tension ranges from ~1% in cartilage to ~13% in major arteries, thus 21% O2 represents a supraphysiologic O2 exposure. We conducted experiments to establish and characterize hUVEC at 21% O2 and physiologically relevant oxygen tension (5% O2) from three independent donors.hUVEC were isolated by standard methods with dissociation and culture steps conducted at 21% or 5% O2. Cells were maintained in standard endothelial cell growth media and were cryopreserved at the end of passage 1. Cells were thawed, used to establish live passage 2 cultures, and confirmed to be sterile and free of mycoplasma and endotoxin. hUVEC established at 5% O2 (hUVEC‐LO) were compared to hUVEC established at 21% O2 (hUVEC‐HO) with respect to population doubling time, surface marker expression by flow cytometry, and angiogenic potential by tube formation assay.Both hUVEC‐LO and hUVEC‐HO were immunopositive for CD31, VE‐cadherin, CD146, CD202b, and vEGFR1, and were negative for CD45 and CD90. hUVEC‐LO uniquely expressed a greater percentage of markers associated with an endothelial progenitor phenotype, including CD117, Slug, CD133, and vEGFR2. The calculated population doubling time of hUVEC‐LO was on average 10% faster than the donor‐matched hUVEC‐HO. Angiogenic potential was confirmed by tube formation assay for each lot at the oxygen tension at which they were established. Crossover experiments were conducted to assess tube formation in hUVEC‐LO and hUVEC‐HO under assay conditions of 21% or 5% O2. No statistical differences were noted after 6 hours of assay duration with respect to: number of junctions, segments, meshes, and total length of segments, branches, or extremities. Significant differences were observed beyond 6 hours between hUVEC‐LO and hUVEC‐HO in mean mesh size and number of branches, but the magnitude and pattern of differences varied from donor‐to‐donor. In contrast, the early phase of angiogenesis (0–6 hours) revealed notable differences between hUVEC‐LO and hUVEC‐HO, with hUVEC‐LO generally exhibiting greater variance with respect to number of junctions, mesh area, number of meshes, and number of segments.Taken together, these results suggest that hUVEC‐LO may have a more stem‐like phenotype characterized by the retention of progenitor‐associated surface markers and a faster doubling time compared to hUVEC‐HO from the same donor. While the ultimate capacity for tube formation did not differ between hUVEC‐LO and hUVEC‐HO assayed at 21% or 5% O2, the preliminary data suggest that hUVEC‐LO respond more actively to adapt to changes in oxygen tension, undergoing significant fluctuations in multiple measures at early time points. Further experiments are needed to elucidate the processes and mechanism(s) that lead to differential phenotype and/or performance of hUVEC‐LO and hUVEC‐HO, in particular during the early initiating events of angiogenesis.

Primary Endothelial Cells from Seals Mount a Rapid and Sustained Response to Acute Hypoxia

Marine mammals tolerate repeated bouts of hypoxemia while diving, with up to 90% of blood oxygen stores depleted in some species. Accordingly, tissues such as the endothelium experience acute fluctuations in oxygen availability. In terrestrial mammals, hypoxia/reoxygenation events of similar magnitude contribute to inflammatory signaling, reactive oxygen species (ROS) generation, and endothelial dysfunction in pathological settings. Marine mammals, however, appear well protected against oxidant stress and inflammation despite repeated exposure to hypoxia/reoxygenation, though the specific cellular mechanisms driving this protection remain unknown. We isolated endothelial cells (ECs) from placental arteries from northern elephant seals (Mirounga angustirostris), a pinniped species noted for its extreme diving capacity. Seal primary EC cultures proliferate, express the canonical EC markers PECAM‐1 and VE‐cadherin, and take up acetylated LDL. Seal ECs are metabolically active in adherent culture and respond to mitochondrial inhibition and uncoupling and pharmacological NADPH oxidase activation, demonstrating utility as a live, functionally relevant system in which to study the biochemical drivers of hypoxia/reoxygenation tolerance in diving seals. Seal ECs exposed to 1% O 2 for up to 6 hours upregulated HIF‐1α protein, with maximal expression occurring within 15 minutes (68‐fold increase over normoxic levels). Human ECs matched this magnitude (73‐fold increase) though not until 1 hour, after which expression of HIF‐1α rapidly and progressively declined (22‐fold above normoxic levels at 6 hours). In contrast, seal ECs maintained elevated HIF‐1α protein expression (52‐fold) at 6 hours. Pharmacological stimulation with menadione, an inducer of redox cycling, significantly increased oxidant generation in human (5‐fold, p=0.027) but not seal ECs (2.5‐fold, p=0.18), as did acute exposure to hypoxia/reoxygenation. Together, our data show that seal ECs mount a rapid, sustained response to hypoxia /reoxygenation events, and that these cells are a functional system in which to study the mechanisms underlying innate hypoxia and oxidative stress tolerance in diving mammals.Support or Funding InformationNational Science Foundation GRFP

Aqueous humor induces lymphatic regression on the ocular surface

PurposeThis study is to investigate the potential effect of aqueous humor on already formed lymphatic vessels of the ocular surface including the conjunctiva and the cornea. MethodsAqueous humor harvested from fresh bovine or murine eyeballs were used in the study. It was injected into the subconjunctival space of Prox-1-GFP (green fluorescent protein) transgenic mice. Pre-existing conjunctival lymphatics were observed in vivo using our advanced live imaging system. Additionally, ex vivo tissue cultures were performed in aqueous humor with normal conjunctival tissues or inflamed corneas with newly formed lymphatic vessels. Time lapse images were taken by an advanced live cell imaging system with an incubator. Moreover, human primary microdermal lymphatic endothelial cell culture system was employed to evaluate the effect of aqueous humor on lymphatic tube regression in vitro. ResultsAqueous humor induced lymphatic regression in both normal conjunctiva and inflamed corneas. It also led to the regression of formed lymphatic tubes by the lymphatic endothelial cells in vitro. ConclusionsThis study provides the first direct and real time live imaging evidence showing that aqueous humor induces lymphatic regression. Further investigation promises for divulging new mechanisms and therapeutic strategies to treat lymphatic diseases that occur both inside and outside the eye.

Endothelium-Targeted Deletion of microRNA-15a/16-1 Promotes Poststroke Angiogenesis and Improves Long-Term Neurological Recovery.

Angiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. However, the function of the miR-15a/16-1 cluster in endothelium on postischemic cerebral angiogenesis is not known. To investigate the functional significance and molecular mechanism of endothelial miR-15a/16-1 cluster on angiogenesis in the ischemic brain. Endothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice and wild-type littermate controls were subjected to 1 hour middle cerebral artery occlusion followed by 28-day reperfusion. Deletion of miR-15a/16-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/16-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/16-1 cluster up-regulated the protein expression of pro-angiogenic factors VEGFA (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2), and their receptors VEGFR2 (vascular endothelial growth factor receptor 2) and FGFR1 (fibroblast growth factor receptor 1) after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/16-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation, whereas lentiviral overexpression of the miR-15a/16-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/16-1 translationally represses pro-angiogenic factors VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within 3'-untranslated regions of those messenger RNAs. Endothelial miR-15a/16-1 cluster is a negative regulator for postischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/16-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

A dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation

The blood-brain barrier (BBB) serves to protect and regulate the CNS microenvironment. The development of an in-vitro mimic of the BBB requires recapitulating the correct phenotype of the in-vivo BBB, particularly for drug permeation studies. However the majority of widely used BBB models demonstrate low transendothelial electrical resistance (TEER) and poor BBB phenotype. The application of shear stress is known to enhance tight junction formation and hence improve the barrier function. We utilised a high TEER primary porcine brain microvascular endothelial cell (PBMEC) culture to assess the impact of shear stress on barrier formation using the Kirkstall QuasiVivo 600 (QV600) multi-chamber perfusion system. The application of shear stress resulted in a reorientation and enhancement of tight junction formation on both coverslip and permeable inserts, in addition to enhancing and maintaining TEER for longer, when compared to static conditions. Furthermore, the functional consequences of this was demonstrated with the reduction in flux of mitoxantrone across PBMEC monolayers. The QV600 perfusion system may service as a viable tool to enhance and maintain the high TEER PBMEC system for use in in-vitro BBB models.

Efficient isolation and identification of primary endothelial cells from bovine aorta by collagenase P

Introduction: Endothelial cells are widely used among researchers for investigation of cardiovascular diseases, particularly atherosclerosis. Since aortic endothelial cells are able to be cultured in high passages, these cells are suitable for physiological and pathological studies of blood vessels. Objectives: The aim of this study was employing a digestion method to isolate the endothelial cells from bovine aorta by utilizing collagenase P and, establishing and characterizing isolated primary endothelial cells (IPECs) cultures. Materials and Methods: IPECs were isolated from fresh bovine aorta via enzymatic digestion method using collagenase P. Cell morphology and its functions were assessed by measuring the gene expression of endothelial nitric-oxide synthase (eNOS) and endothelin-1. In order to validate them as IPECs, they were compared with bovine aortic endothelial cells (BAECs) and vascular smooth muscle cells (VSMCs). Effects of synthetic endothelin-1 (100 nm) were assessed on the phosphorylation of Smad2 transcription factor via western blotting in IPECs for periods of one to four hours. Results: In this study, the morphology of IPECs from bovine aorta was identical to that of the BAECs. The gene expressions of endothelin-1 and eNOS were higher than those of BAECs and VSMCs. In addition, synthetic endothelin-1 resulted in the time-dependent boost of phosphorylation of carboxy-terminal Smad2 in the IPECs for periods of two and four hours. Conclusion: The results of this study confirm the efficacy of the enzymatic digestion method in isolating a large number of endothelial cells with morphological and functional characteristics.