Primary Cultures Of Endothelial Cells Research Articles

Shear stress facilitates Akt activation in human pulmonary microvascular endothelial cells

Introduction: Pulmonary arterial obstruction due to acute thromboembolism causes local reduction or cessation of blood flow. Under normal conditions, pulmonary blood flow exerts shear stress on endothelial cells, known to play an important role in maintaining vascular homeostasis. We hypothesized that pulmonary microvascular endothelial cells exposed to abrupt reductions in shear stress would demonstrate attenuated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and phosphorylation of downstream effectors Akt and endothelial nitric oxide synthase (eNOS). Methods: We tested our hypothesis in primary cultures of human lung microvascular endothelial cells (hLMVECs). We cultured early passage (p≤8) hLMVECs from 5 unique donor lots in standard 6-well culture plates and exposed cells to shear stress using an orbital shaker. Using a fixed depth (volume) and viscosity of tissue culture media, we adjusted the magnitude of shear stress exerted on hLMVECs by changing the angular velocity of orbital rotation. We acclimatized hLMVECs to physiologically-relevant shear stress (~12 dyn/cm 2 ) for 24 hours, then exposed cells to low shear stress (~3 dyn/cm 2 ) or no shear stress (0 dyn/cm 2 ) for 30 minutes. Control cells were maintained at 12 dyn/cm 2 of shear stress. In some experiments, PI3K activity was inhibited throughout the acclimatization and shear stress manipulation period by including LY294002 (10 μM) or vehicle (DMSO, 0.1% v/v) in culture media. We prepared whole cell lysates and used western blot to evaluate the expression and phosphorylation of Akt (ser473) and eNOS (ser1177). Results: We found that phosphorylation of Akt, but not Akt expression, decreased significantly (p<0.05) when shear stress was acutely reduced (~3 dyn/cm 2 ) or removed (0 dyn/cm 2 ). Inhibition of PI3K activity with LY294002 further diminished shear-mediated changes in Akt phosphorylation in all 3 groups. Conversely, eNOS phosphorylation was not modified by acute reductions in shear stress, but eNOS expression was markedly attenuated by PI3K inhibition under all conditions. Conclusions: Our results indicate that phosphorylation of Akt, but not its downstream target eNOS, is rapidly attenuated by abrupt reductions in shear stress in hLMVECs. However, shear stress appears to regulate eNOS expression in a PI3K-dependent manner. Taken together, these findings suggest that acute disruption of flow, as seen in pulmonary embolism, may disrupt human lung microvascular endothelial cell homeostasis and nitric oxide bioavailability via multiple PI3K-dependent signaling pathways. This work is supported by HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Precursors of specialized pro-resolving mediators (SPMs) are decreased in hypertension, and treatment with SPMs improve vascular relaxation via formyl peptide-receptor-2

Specialized pro-resolving mediators (SPMs), derived from essential fatty acids, play an immunoregulatory role. Formyl peptide receptor-1 is a pattern recognition receptor that mediate host-defense and inflammation. On the other hand, FPR2 binds SPMs to induce resolution of inflammation. It is well known that low-grade chronic inflammation is present in hypertension, but it is unknown whether this phenomenon occurs due to dysregulation of resolution of inflammation. Also, it is unclear if arteries can sense SPM. We hypothesized that the resolution of inflammation is decreased in hypertension, and treatment with SPM will improve vascular function via FPR-2. Forty-five eicosanoids, precursors of SPMs were detected in the plasma of male Wistar Kyoto rats (WKY) (14 weeks old, n=10) and spontaneously hypertensive rats (SHR; 14 weeks old, n=16). Analysis revealed a significant decrease (%) in 5 different eicosanoids in SHR (1) free eicosapentaenoic acid (60%), (2) free adrenic acid (25%), (3) 20-carboxy-arachidonic acid (52%) (4) 5-hydroxyeicosatetraenoic acid (20%), and (5) 14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) (48%). Additionally, mesenteric resistance arteries (MRA, lumen diameter < 300 μm) from SHR rats showed significant oxidative degradation of lipids measured by the presence of 4-hydroxynonenal (4-HNE) (4-HNE; AU: WKY: 3.8±0.6 vs. SHR: 14.4±3.8; p=0.02). MRA were mounted in a wire myograph to evaluate vascular function, and concentration-response curves (CRC) to acetylcholine (ACh 10−9 to 10−5 mol/L) were performed. As expected, relaxation was impaired in MRA from SHR compared to WKY. This was noted by a shift to the right in the CRC, increasing (%) area under the curve (ΔAUC: 20± 4%). On the other hand, SPMs (10 nM) restored this response by shifting the CRC to the left and reducing the AUC compared to arteries from SHR treated with vehicle [SPMs: Resolvin D1 (RvD1) (ΔAUC:19±4%), Resolvin E1 (RvE1) (ΔAUC: 8.5 ± 9%), and lipoxin A4 (ΔAUC: 20±7%,)]. Specific antagonism of FPR-2 (WRW4, 1μM) abolished the improvement in relaxation induced by SPMs. Superoxide indicator dihydroethidium (DHE) was used in primary MRA endothelial cell culture. SPMs reduced the formation of reactive oxygen species after an inflammatory stimulus [Control: 99±2.5%; TNFα: 272± 50%; RvD1: 109±25%; RvE1: 111±22%; LXA4: 101±10%, A.U.). However, WRW4 did not change these responses. Total FPR2 and lipoxygenase 5 (LOX-5) protein expression was similar in arteries from WKY and SHR. However, there was an increase in LOX-5 serine 523 phosphorylation in SHR, suggesting a compensatory mechanism to increase local LXA4. Overall, SPMs play an important role to maintain vascular heath, and their effects occur both dependent and independent of FPR2 activation. Improvement of resolution of inflammation maybe a new treatment for for hypertension. National Institutes of Health - R00GM118885, R01HL149762, R00HL151889, and RO1DK132948. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PRIMARY CULTURE AND IDENTIFICATION OF MOUSE BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Brain microvascular endothelial cells are the basic components of the blood-brain barrier. Many neurological diseases are related to the loss of blood brain barrier (BBB) function. Isolating and culturing primary BMVEC is an important means to study the function and regulation of BBB in vitro. To establish a method for isolation and culture of primary mouse brain microvascular endothelial cells (BMECs). The 7-10-days mice were sacrificed by neck removal, the cranial cavity was opened, the brain was aseptically removed and the cerebral cortex was retained. To extract brain microvascular segments, the brain underwent three D-Hank solution rinses. It was then homogenized, twice digested by enzyme, and centrifuged using a Bovine Serum Albumin (BSA) gradient. For primary culture, the brain microvascular segments were injected into gelatin-coated culture plates using DMED complete media. When the cell density reaches 90 %, the media is removed, and the cells are then given two PBS washes. A new medium was introduced after adding 1ml of the trypsin-EDTA solution to digest for 2–5 minutes for passage. Cell culture plates were rinsed and pre-cooled 95 % ethanol was added for 20 minutes after passaged cells had grown to 80–90 % of their original size. After a third wash, 1 ml of mouse factor VIII antibody was added to the culture wells, where it was left for 4 hours at 37 °C. FITC-labeled rabbit anti-mouse antibody in a volume of 1 mL was added. Under a confocal laser microscope, the plates were examined and taken pictures of. The outcome demonstrates positive expression of the marker factor VIII associated antigen. To create a cell suspension, monolayer-growing microvascular endothelial cells were chosen. 100 L of cell suspension was used to inoculate each well in 96-well plates, and the cells were then grown. On days 1, 2, 3, 4, 5, 6, and 7, the original medium was removed, and then 180 μL of DMEM and 20 μL of MTT were simultaneously added to each well for a 4-hour culture. After adding dimethyl sulfoxide (DMSO), each well's absorbance value (A value) was measured at 492 nm. The outcome demonstrates that the growth peak is attained between 6 and 8 days. This method can successfully isolate and culture primary BMECs, which lay a foundation for the study of BMEC in vitro.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

Antiretroviral drugs efavirenz, dolutegravir and bictegravir dysregulate blood-brain barrier integrity and function.

The implementation of combined antiretroviral therapy (cART) significantly reduces the mortality associated with human immunodeficiency virus (HIV) infection. However, complications such as HIV-associated neurocognitive disorders (HAND) remain a major health concern. We hypothesized that the toxicity of antiretroviral drugs (ARVs) may contribute to the pathogenesis of HAND in addition to cerebral viral infection. To address this question, we evaluated the impact of HIV integrase strand transfer inhibitors (dolutegravir and bictegravir), and a non-nucleoside reverse transcriptase inhibitor (efavirenz) on the integrity and permeability of various human and mouse blood-brain barrier (BBB) models, in vitro, ex vivo and in vivo. We observed a significant downregulation of tight junction proteins (TJP1/Tjp1, OCLN/Ocln and CLDN5/Cldn5), upregulation of proinflammatory cytokines (IL6/Il6, IL8/Il8, IL1β/Il1β) and NOS2/Nos2, and alteration of membrane-associated transporters (ABCB1/Abcb1a, ABCG2/Abcg2 and SLC2A1/Slc2a1) mRNA expression, in vitro, in human (hCMEC/D3) and primary cultures of mouse microvascular endothelial cells, and ex vivo in isolated mouse brain capillaries treated with efavirenz, dolutegravir, and/or bictegravir. We also observed a significant increase in BBB permeability in vivo following treatment with the selected ARVs in mice applying NaF permeability assay. Taken together, these results suggest that clinically recommended integrase strand transfer inhibitors such as dolutegravir may exacerbate HIV-associated cerebrovascular pathology, which may contribute to the associated short-term neuropsychiatric side effects and the high incidence of mild forms of HAND reported in the clinical setting.

Aging decreases docosahexaenoic acid transport across the blood-brain barrier in C57BL/6J mice.

Nutrients are actively taken up by the brain via various transporters at the blood-brain barrier (BBB). A lack of specific nutrients in the aged brain, including decreased levels of docosahexaenoic acid (DHA), is associated with memory and cognitive dysfunction. To compensate for decreased brain DHA, orally supplied DHA must be transported from the circulating blood to the brain across the BBB through transport carriers, including major facilitator superfamily domain-containing protein 2a (MFSD2A) and fatty acid-binding protein 5 (FABP5) that transport esterified and non-esterified DHA, respectively. Although it is known that the integrity of the BBB is altered during aging, the impact of aging on DHA transport across the BBB has not been fully elucidated. We used 2-, 8-, 12-, and 24-month-old male C57BL/6 mice to evaluate brain uptake of [14C]DHA, as the non-esterified form, using an in situ transcardiac brain perfusion technique. Primary culture of rat brain endothelial cells (RBECs) was used to evaluate the effect of siRNA-mediated MFSD2A knockdown on cellular uptake of [14C]DHA. We observed that the 12- and 24-month-old mice exhibited significant reductions in brain uptake of [14C]DHA and decreased MFSD2A protein expression in the brain microvasculature compared with that of the 2-month-old mice; nevertheless, FABP5 protein expression was up-regulated with age. Brain uptake of [14C]DHA was inhibited by excess unlabeled DHA in 2-month-old mice. Transfection of MFSD2A siRNA into RBECs decreased the MFSD2A protein expression levels by 30% and reduced cellular uptake of [14C]DHA by 20%. These results suggest that MFSD2A is involved in non-esterified DHA transport at the BBB. Therefore, the decreased DHA transport across the BBB that occurs with aging could be due to age-related down-regulation of MFSD2A rather than FABP5.

Adjusting Degree of Modification and Composition of gelAGE-Based Hydrogels Improves Long-Term Survival and Function of Primary Human Fibroblasts and Endothelial Cells in 3D Cultures.

This study aimed to develop a suitable hydrogel-based 3D platform to support long-term culture of primary endothelial cells (ECs) and fibroblasts. Two hydrogel systems based on allyl-modified gelatin (gelAGE), G1MM and G2LH, were cross-linked via thiol-ene click reaction with a four-arm thiolated polyethylene glycol (PEG-4-SH). Compared to G1MM, the G2LH hydrogel was characterized by the lower polymer content and cross-linking density with a softer matrix and homogeneous and open porosity. Cell viability in both hydrogels was comparable, although the G2LH-based platform supported better F-actin organization, cell-cell interactions, and collagen and fibronectin production. In co-cultures, early morphogenesis leading to tubular-like structures was observed within 2 weeks. Migration of fibroblasts out of spheroids embedded in the G2LH hydrogels started after 5 days of incubation. Taken together, the results demonstrated that the G2LH hydrogel fulfilled the demands of both ECs and fibroblasts to enable long-term culture and matrix remodeling.

Abstract TP18: Iron Overload Induces Cognitive Deficits And Autophagy Impairment In The Brains Of Aged Mice In A Sex-Dependent Manner

Iron deposits have been observed in postmortem brain samples from Alzheimer’s disease patients. Different iron overload models have been used to study neurodegeneration in young mice. However, the effects of excess iron with aging, which is a critical factor for Alzheimer’s disease and iron deposition in the brain, remain unexplored. In addition, iron metabolism is regulated by autophagy, a cellular process that degrades intracellular components. Brain autophagy is downregulated with aging and in females, and autophagy impairment occurs in many neurodegenerative disorders. Thus, we hypothesized that sex differences exist in autophagy in the brains of aged mice treated with iron overload. We treated aged (18-20 months) WT mice of both sexes with iron overload (5% ferric citrate, FC, 3 days/week/6 weeks), or a vehicle, by oral gavage. We found that aged females showed impairment in conditioning memory (Fear conditioning), but not aged males. Aged females treated with FC also showed significant increase of iron deposition in their brains, compared with control females. However, we did not find significant differences between control and treated male mice. Furthermore, the brain lysates of aged females treated with iron overload showed increased levels of the early autophagy marker BECLIN1 (in both sexes), and enhanced levels of the autophagy cargo p62 (in both sexes) and the lysosomal marker LAMP1 (only in females), compared with control mice. This suggests that iron overload induces the initial stage of autophagy in both sexes, but impairs late autophagy, mainly in female mice, compared with control mice. Furthermore, in primary culture of cerebral endothelial cells (CEC) isolated from aged mice, we found that CEC treated with ferric citrate exhibit enhanced DNA damage and reduced cell proliferation in a dose-dependent manner. Importantly, CEC derived from female mice showed reduced autophagy flux, compared with male CEC. This suggests that iron overload may be more detrimental in the cerebrovasculature of aged female mice than in males, and this could explain iron overload-induced memory deficiency in aged female mice, but not in males.

Nanomolar clodronate induces adenosine accumulation in the perfused rat mesenteric bed and mesentery-derived endothelial cells.

The vesicular nucleotide transporter (VNUT) is critical for sympathetic co-transmission and purinergic transmission maintenance. To examine this proposal, we assessed whether the bisphosphonate clodronate, claimed as a potent in vitro VNUT blocker, modified spontaneous and/or the electrically evoked overflow of ATP/metabolites and NA from mesentery sympathetic perivascular nerve terminals. Additionally, in primary endothelial cell cultures derived from this tissue, we also evaluated whether clodronate interfered with ATP/metabolite cell outflow and metabolism of N6-etheno adenosine 5'-triphosphate (eATP), N6-etheno adenosine (eADO), and adenosine deaminase enzyme activity. Rat mesenteries were perfused in the absence or presence of .01-1,000nM clodronate, 1-1,000nM Evans blue (EB), and 1-10µM DIDS; tissue perfusates were collected to determine ATP/metabolites and NA before, during, and after perivascular electrical nerve terminal depolarization. An amount of 1-1,000nM clodronate did not modify the time course of ATP or NA overflow elicited by nerve terminal depolarization, and only 10nM clodronate significantly augmented perfusate adenosine. Electrical nerve terminal stimulation increased tissue perfusion pressure that was significantly reduced only by 10nM clodronate [90.0 ± 18.6 (n = 8) to 35.0 ± 10.4 (n = 7), p = .0277]. As controls, EB, DIDS, or reserpine treatment reduced the overflow of ATP/metabolites and NA in a concentration-dependent manner elicited by nerve terminal depolarization. Moreover, mechanical stimulation of primary endothelial cell cultures from the rat mesentery added with 10 or 100nM clodronate increased adenosine in the cell media. eATP was metabolized by endothelial cells to the same extent with and without 1-1,000nM clodronate, suggesting the bisphosphonate did not interfere with nucleotide ectoenzyme metabolism. In contrast, extracellular eADO remained intact, indicating that this nucleoside is neither metabolized nor transported intracellularly. Furthermore, only 10nM clodronate inhibited (15.5%) adenosine metabolism to inosine in endothelial cells as well as in a commercial crude adenosine deaminase enzyme preparation (12.7%), and both effects proved the significance (p < .05). Altogether, present data allow inferring that clodronate inhibits adenosine deaminase activity in isolated endothelial cells as in a crude extract preparation, a finding that may account for adenosine accumulation following clodronate mesentery perfusion.

Regenerative effects of mouse aortic endothelial cells in a murine model of critical limb ischemia

Critical limb ischemia of the is a serious disease that threatens a significant decrease in working ability and disability of patients. Cell therapy may be useful in correcting the endothelial dysfunction that accompanies this disorder. The aim of study was to evaluate the effectiveness of local transplantation of mouse aortic endothelial cells (MAECs) in a model of critical limb ischemia in mice. Materials and methods. Critical limb ischemia in FVB mice was modeled by femoral artery ligation. The primary culture of endothelial cells was obtained from the murine aortic intima. The endothelial phenotype of cells for the expression of CD31, CD38 and CD309 markers was confirmed by flow cytometry and 1•106 MAECs were transplanted intramuscularly into ischemic limb. Tissue perfusion was assessed by laser Doppler flowmetry as well as descriptive histology was used to analyze changes in ischemic muscle after cell transplantation compared to the control group. Results. After MAECs transplantation in animals with modeled critical limb ischemia, the skin of the foot kept pink color and the corresponding temperature of the healthy limb without signs of necrosis of the distal phalanges in contrast to animals of the control group. According to laser Doppler flowmetry data, a significant difference (p ≤ 0.05) in perfusion of ischemic and sham-operated limbs in animals of the control group remained at the level of Δ = 45.7 ± 13.1 %. In animals after MAECs transplantation, the difference of these indicators between limbs was only Δ = 14.0 ± 8.23 % and was not statistically significant. A histological examination of muscle tissue after MAECs transplantation demonstrated the signs of compensatory processes characterized by hyperplasia and hypertrophy of myocyte’s nuclei and lightening of the nucleoplasm with well-defined nucleoli in some myofibrils. In the cytoplasm of myocytes, intermediate Z-discs were clearly visualized, and the number of myofibrils in muscle fibers increased. Conclusion. In animals with model of critical limb ischemia, the transplantation of aorta-derived endothelial cells recover the perfusion of ischemic limbs and improve the histological indicators of muscle tissue.

Oxidative stress in the endothelial cell culture exposed to mitomycin C

Background. Atherosclerosis is one of the leading cardiovascular pathologies. Evidence suggests that DNA damage caused by endothelial cell exposure to mitomycin C (MMC) leads to endothelial dysfunction and is the risk factor for this disease. MMC is an alkylating mutagen involved in the development of oxidative stress, which is also a risk factor for atherosclerosis. Aim. To access the levels of oxidative stress markers in the primary human endothelial cell culture exposed to alkylating mutagen MMC.Material and Methods. Commercially available primary cultures of endothelial cells obtained from human coronary artery (HCAEC) and human internal thoracic artery (HITAEC) were used in the study. The cells were cultivated in the presence of 500 ng/mL MMC (experimental group) and without mutagenic load (control group). The levels of reactive oxygen species, reactive nitrogen species, and 8-OH-deoxyguanosine (8-OHdG) in cell growth media were assessed by enzyme-linked immunosorbent assay. The relative telomere length and expression of TERT and POT1 genes were accessed in endothelial cells by quantitative polymerase chain reaction. Statistical analysis of data was performed using GraphPad Prism 9 software.Results. There were no differences in the concentrations of reactive oxygen species, reactive nitrogen species (NO2 -, NO3 -, NO2 -/NO3 -), and 8-OHdG in HCAEC and HITAEC cultures exposed to MMC compared to the corresponding parameters in the non-exposed controls. At the same time, HCAEC and HITAEC exposed to MMC were characterized by a decrease in the relative telomere length compared to control (10.97 vs. 27.03 in HCAEC, p = 0.002 and 9.12 vs. 25.64 in HITAEC, p = 0.001). Moreover, we discovered 1.75-fold increase in the expression of POT1 gene in the experimental HCAEC compared to control (p = 0.019). No expression of TERT gene was observed in study groups.Conclusions. Alkylating mutagen MMC did not induce any pronounced oxidative stress in the primary human endothelial cells in vitro. The development of endothelial dysfunction caused by MMC exposure was triggered mainly by DNA alkylation resulting in the genotoxic stress in the endothelial cells.

Enrichment in procoagulant microparticles in calcified human aortic valve – role in valvular endothelium alterations and enhanced thrombogenicity

Abstract Background Aortic stenosis (AS) is characterized by endothelial dysfunction (ED), inflammatory cell infiltration, myofibroblastic and osteoblastic differentiation. Subclinical leaflet thrombosis was recently linked to higher rates of stroke and transient ischemic attack after transcatheter aortic valve implantation (TAVI). Procoagulant microparticles (MPs) are associated with ED, inflammation and clot formation. There is limited evidence regarding intra-valvular MPs content and their potential biological effects. This question is particularly relevant in TAVI in which the residing native valve could constitute a source of thrombotic activity enhancing leaflet thrombosis and valve dysfunction. Purpose Therefore, we hypothesized that MPs trapped within the native aortic valve contribute to valvular dysfunction including enhanced thrombogenicity. Methods Human valves were collected from patients undergoing surgical valve replacement for AS or aortic insufficiency (AI). Pro-thrombotic, pro-inflammatory, and ED markers were identified in the calcified vs non-calcified part of the valves by Western-blot. Calcium content was measured through colorimetric method. MPs were extracted from human pathological valves, and quantified through their prothrombinase activity. Primary cultures of porcine valvular endothelial cells (VEC) were treated with the MPs (10 nmol/L) or thrombin (1U/ml) for 24hrs. Phenotypic change was appreciated through gene expression pattern assessed by RT-qPCR. IL-8 secretion was measured by ELISA. Results The phenotype of the AS valve was characterized through increased expression of thrombogenic (tissue factor, thrombomodulin, PAI-1), adhesive (VCAM-1, ICAM-1) and inflammatory (COX-1, COX-2) molecules in the calcified part of the valve. Moreover, MPs content was increased in the calcified vs non-calcified part of the valve or AI valves. MPs levels was correlated with valvular calcium content (R=0.3862: p&amp;lt;0.001). Tissue factor was increased in MPs extracted from AS vs AI. The biological effect of MPs was tested on VEC in-vitro. Results showed dramatic increase in expression of inflammatory cytokines (CXCL10, CCL11, CXCL8, MCP1) adhesion molecules (VCAM-1, ICAM-1, SELP, SELE) and proangiogenic factors (VEGFR2, ANGPTL4) in VEC exposed to MPs (24h) from AS vs AI. Enhanced secretory phenotype was evidenced through IL-8 determination in the supernatant of VEC stimulated with MPs from AS valve. Conclusion Calcified aortic valve is a potent reservoir of MPs, acting as a pro-thrombogenic source per se and promoting a switch of VEC phenotype toward prothrombotic, proinflammatory and proangiogenic pattern. These data suggest that MPs released from the native valve constitute an important source of mediators involved in enhanced thrombogenicity and valvular remodeling. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): GERCA-Groupe Etudes Reali Commercia Avignon

CLMP Promotes Leukocyte Migration Across Brain Barriers in Multiple Sclerosis.

Background and ObjectivesIn multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor–like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS.MethodsExpression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP.ResultsUsing bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP+ B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP+ immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS.DiscussionCollectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.

HIV-1 gp120 and tobacco smoke synergistically disrupt the integrity of the blood-brain barrier

In the United States, the Centers for Disease Control and Prevention (CDC) terms HIV and tobacco use among the ten most important public health challenges we face today. In the last decade, there has been a remarkable decrease in the number of deaths due to HIV/AIDS, especially after the widespread availability and use of combination antiretroviral therapy (cART). However, people living with HIV/AIDS have a heightened risk of chronic complications and comorbidities, including neurological disorders. Around 40–60 % of HIV-infected individuals progress to NeuroAIDS, a group of disorders caused primarily by HIV-mediated damage to the central and peripheral nervous systems, despite receiving cART. The detrimental effects of chronic smoking on the cerebrovascular system are also well studied and reported. Addictive behavior, such as smoking, is more common in HIV patients compared to the general population. In this context, given the existing immune suppression, smoking can pose a significant risk for the progression of the disease to NeuroAIDS by disrupting the integrity of the blood-brain barrier (BBB). Here we show that co-treatment with Tobacco Smoke Extract (TSE) and HIV-1 gp120 (HIV envelope glycoprotein) in primary cultures of human brain microvascular endothelial cells promoted heightened cellular stress responses compared to control and individual treatments. Our findings suggest that a potential synergistic effect between smoke exposure and gp120 can worsen the loss of BBB viability, possibly exacerbating NeuroAIDS progression.

Isolation and short-term culturing of primary lymphatic endothelial cells from collecting lymphatics: A techniques study.

To develop an experimental method for routine isolation and short-term culture of primary lymphatic endothelial cells from specific collecting vessels. Lymphatic endothelial cell tubes (LECTs) were isolated from micro-dissected collecting vessels. LECTs were allowed to attach and grow for ~3 weeks before being passaged. Non-purified cultures were partially characterized by immunofluorescence and RT-PCR at passages 1-2. The method was validated in cultures of primary lymphatic endothelial cells (LECs) from male and female mice. After 1 or 2 passages, >60% of the LECs maintained expression of Prox1. Expression of 22 different genes was assessed using RT-PCR. Prox1, Vegfr3, eNos, Cdh5, Pecam1, Cx43, Cx37, and Cx47, among others, were expressed in these short-term cultured LECs, while Myh11, Cnn1, Desmin, and Cd11b were not detected. Prox1 expression, as determined by western blotting, was similar in cultured LECs from age-matched male and female mice. Confocal imaging of intracellular calcium in cultures of primary LECs from Cdh5-GCaMP8 mice demonstrated that a functional phenotype was maintained, similar to lymphatic endothelial cells in freshly isolated vessels. This method provides an innovative tool for routine isolation and study of primary LECs from specific collecting lymphatic vessels from any mouse, and in fact, from other species.

Vascular response to stress: Protective action of the bisphosphonate alendronate.

Since several additional actions of bone bisphosphonates have been proposed, we studied the effect of the bisphosphonate alendronate (ALN) on the vascular response to environmental stress. Primary cultures of endothelial cells (EC) and vascular smooth muscle cells (VSMC) exposed to strained conditions were employed for experimental evaluation. After ALN treatment, cell migration, proliferation, and angiogenesis assays were performed. The participation of signal transduction pathways in the biochemical action of ALN was also assessed. In VSMC cultures, ALN counteracted the stimulation of cellular migration elicited by the proinflammatory agent lipopolysaccharide (LPS) or by high levels of calcium and phosphorus (osteogenic medium). Indeed, ALN reduced the increase of VSMC proliferation evoked by the stressors. When LPS and osteogenic medium were added simultaneously, the enhancement of cell proliferation dropped to control values in the presence of ALN. The mechanism of action of ALN involved the participation of nitric oxide synthase, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways. The study revealed that ALN exhibits a proangiogenic action. On EC, ALN enhanced vascular endothelial growth factor (VEGF) synthesis, and induced capillary-like tube formation in a VEGF-dependent manner. The presence of vascular stress conditions (LPS or osteogenic medium) did not modify the proangiogenic action elicited by ALN. The findings presented suggest an extra-bone biological action of ALN, which could contribute to the maintenance of vascular homeostasis avoiding cellular damage elicited by environmental stress.

Alisporivir Normalizes Mitochondrial Function of Primary Mouse Lung Endothelial Cells Under Conditions of Hyperglycemia

Effect of alisporivir (a mitochondrial permeability transition pore inhibitor) on the development of mitochondrial dysfunction under hyperglycemic conditions in the primary culture of mouse lung endothelial cells was investigated in this work. We demonstrated that hyperglycemia (30 mM glucose for 24 h) leads to the decrease in viability of the pulmonary endotheliocytes, causes mitochondrial dysfunction manifested by the drop in membrane potential and increase in superoxide anion generation as well as facilitates opening of the mitochondrial permeability transition pore (MPT pore). Incubation of endothelial cells with 5 µM alisporivir under hyperglycemic conditions leads to the increase in cell viability, restoration of the membrane potential level and of the MPT pore opening activity to control values. Hyperglycemia causes increased mitophagy in the lung endothelial cells: we observed increase in the degree of colocalization of mitochondria and lysosomes and upregulation of the Parkin gene expression. Alisporivir restores these parameters back to the levels observed in the control cells. Hyperglycemia results in the increase in the expression of the Drp1 gene in endotheliocytes responsible for synthesis of the protein involved in the process of mitochondria fission. Alisporivir does not significantly alter expression of the genes. The paper discusses mechanisms of the effect of alisporivir on mitochondrial dysfunction in murine pulmonary endotheliocytes under conditions of hyperglycemia.Electronic supplementary materialThe online version contains supplementary material available at 10.1134/S0006297922070033.

Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis.

In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm2. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.

3,4',5-Trimethoxy-trans-stilbene Alleviates Endothelial Dysfunction in Diabetic and Obese Mice via Activation of the AMPK/SIRT1/eNOS Pathway.

3,4′,5-trimethoxy-trans-stilbene (TMS) is a methoxylated derivative of resveratrol. Previous studies showed the vaso-protective effects of resveratrol; nevertheless, research on this derivative is scarce. The current study aimed to explore whether TMS can alleviate endothelial dysfunction in diabetic and obese mice, along with the underlying mechanisms. Thoracic aortas isolated from male C57BL/6J mice and primary cultures of rat aortic endothelial cells were treated with high glucose with or without TMS. High glucose exposure impaired acetylcholine-induced endothelium-dependent relaxations, down-regulated NO bioavailability and the AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1)/endothelial nitric oxide synthase (eNOS) pathway, increased endoplasmic reticulum (ER) stress and oxidative stress, which were reversed by TMS treatment. Moreover, the protective effects of TMS were abolished by Compound C (AMPK inhibitor), and EX527 (SIRT1 inhibitor). The mice were fed with high-fat diet (60% kcal% fat) for 14 weeks to establish a diabetic and obese model, and were orally administered TMS (10 mg/kg/day) in the last 4 weeks. Chronic TMS treatment alleviated endothelial dysfunction via enhancing the AMPK/SIRT1/eNOS pathway and attenuated oxidative stress and ER stress in aortas of diet-induced obese mice. In summary, our study reveals the potent vaso-protective effect of TMS and its therapeutic potential against endothelial dysfunction in metabolic disorders.

Hydroxyapatite/tricalcium phosphate (HA/beta-TCP) scaffold combined with bone and endothelial cells as a potential candidate for oral and maxillofacial bone regeneration.

Cell-based scaffolds have been proposed as useful for tissue repair and bone regeneration in oral and maxillofacial tissue engineering. However, vascularization, a key player in bone regeneration, is often inadequate. To improve this, the use of biphasic calcium phosphate biomaterial (30% hydroxyapatite (HA)-70% tricalcium phosphate (TCP) mixture) HA/beta-TCP-scaffold with defined micron-channel sizes and pore sizes capable of driving angiogenesis, may prove helpful. Therefore, our study aimed to combine the HA/beta-TCP scaffold with in vitro differentiated cells, especially endothelial cells, well-known players in angiogenesis. The HA/beta-TCP-scaffold was combined with primary cultures of human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs). Cells were co-cultured with HA/beta-TCP-scaffold in normoxia (20% oxygen tension) for 14 days. Then, we evaluated: cell adhesion/morphology (Scanning Electron Microscopy and Confocal Microscopy); cell viability (Calcein AM/Propidium Iodide Assay); the presence of multinucleated hOCs (Tartrate Resistant- Acid Phosphatase assay); expression of specific markers of both osteo- and vasculogenic differentiation (Immunohistochemistry). All cell types were viable and well adhered to the entire scaffold surface. Interestingly, bone matrix formation and newly formed vessel-like structures were both detected. Although further investigation is needed, our data proved that differentiated cells combined in vitro with HA/beta-TCP-scaffold prior to implantation could represent a useful strategy for future applications in bone regeneration. Especially, the use of customized micro-vascularized cell-based scaffold would fall within the innovative "personalized medicine” approach.