The endosomal system of primary human vascular endothelial cells and albumin-FcRn trafficking.

Human serum albumin (HSA) has a long circulatory half-life owing, in part, to interaction with the neonatal Fc receptor (FcRn or FCGRT) in acidic endosomes and recycling of internalised albumin. Vascular endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about endocytic trafficking of FcRn-albumin complexes in primary human endothelial cells. To investigate FcRn-albumin trafficking in physiologically relevant endothelial cells, we generated primary human vascular endothelial cell lines from blood endothelial precursors, known as blood outgrowth endothelial cells (BOECs). We mapped the endosomal system in BOECs and showed that BOECs efficiently internalise fluorescently labelled HSA predominantly by fluid-phase macropinocytosis. Pulse-chase studies revealed that intracellular HSA molecules co-localised with FcRn in acidic endosomal structures and that the wildtype HSA, but not the non-FcRn-binding HSAH464Q mutant, was excluded from late endosomes and/or lysosomes. Live imaging revealed that HSA is partitioned into FcRn-positive tubules derived from maturing macropinosomes, which are then transported towards the plasma membrane. These findings identify the FcRn-albumin trafficking pathway in primary vascular endothelial cells, relevant to albumin homeostasis.

Molecular and Cellular Characterization of Primary Endothelial Cells from a Familial Cavernomatosis Patient.

Cerebral cavernous malformation (CCM) or familial cavernomatosis is a rare, autosomal dominant, inherited disease characterized by the presence of vascular malformations consisting of blood vessels with an abnormal structure in the form of clusters. Based on the altered gene (CCM1/Krit1, CCM2, CCM3) and its origin (spontaneous or familial), different types of this disease can be found. In this work we have isolated and cultivated primary endothelial cells (ECs) from peripheral blood of a type 1 CCM patient. Differential functional and gene expression profiles of these cells were analyzed and compared to primary ECs from a healthy donor. The mutation of the familial index case consisted of a heterozygous point mutation in the position +1 splicing consensus between exons 15 and 16, causing failure in RNA processing and in the final protein. Furthermore, gene expression analysis by quantitative PCR revealed a decreased expression of genes involved in intercellular junction formation, angiogenesis, and vascular homeostasis. Cell biology analysis showed that CCM1 ECs were impaired in angiogenesis and cell migration. Taken together, the results obtained suggest that the alterations found in CCM1 ECs are already present in the heterozygous condition, suffering from vascular impairment and somewhat predisposed to vascular damage.

MasR and pGCA receptor activation protects primary vascular smooth muscle cells and endothelial cells against oxidative stress via inhibition of intracellular calcium.

Cardiovascular diseases (CVDs) are associated with vascular smooth muscle cell (VSMC) and endothelial cell (EC) damage. Angiotensin1-7 (Ang1-7) and B-type natriuretic peptide (BNP) are responsible for vasodilation and regulation of blood flow. These protective effects of BNP are primarily mediated by the activation of sGCs/cGMP/cGKI pathway. Conversely, Ang1-7 inhibits Angiotensin II-induced contraction and oxidative stress via Mas receptor activation. Thus, the aim of the study was to determine the effect of co-activation of MasR and particulate guanylate cyclase receptor (pGCA) pathways by synthesized novel peptide (NP) in oxidative stress-induced VSMCs and ECs. MTT and Griess reagent assay kits were used for the standardization of the oxidative stress (H2 O2 ) induced model in VSMCs. The expression of targeted receptors in VSMC was done by RT-PCR and Western blot analysis. Protective effect of NP in VSMC and EC was determined by immunocytochemistry, FACS analysis, and Western blot analysis. Underlying mechanisms of EC-dependent VSMC relaxation were done by determining downstream mRNA gene expression and intracellular calcium imaging of cells. Synthesized NP significantly improved oxidative stress-induced injury in VSMCs. Remarkably, the actions of NP were superior to that of the Ang1-7 and BNP alone. Further, a mechanistic study in VSMC and EC suggested the involvement of upstream mediators of calcium inhibition for the therapeutic effect. NP is reported to possess vascular protective activities and is also involved in the improvement of endothelial damage. Moreover, it is highly effective than that of individual peptides BNP and Ang1-7 and therefore it may represent a promising strategy for CVDs.

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

OBJECTIVE: To develop an experimental method for routine isolation and short-term culture of primary lymphatic endothelial cells from specific collecting vessels. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Furin inhibition prevents hypoxic and TGFß-mediated blood-brain barrier disruption.

Hypoxic blood-brain barrier (BBB) dysfunction is a common feature of CNS diseases however mechanisms underlying barrier disturbance are still largely unknown. This study investigated the role of transforming growth factor ß (TGFß), a cytokine known to induce expression of the proprotein convertase Furin, in hypoxia-mediated barrier compromise. We show that exposure of brain endothelial cells (ECs) to hypoxia (1% O2) rapidly stimulates their migration. Additional exogenous TGFß (0.4 nM) exposure potentiated this effect and increased Furin expression in a TGFß type I receptor activin-like kinase 5 (ALK5) - dependent manner (prevented by 10 µM SB431542). Furin inhibition prevented hypoxia-induced EC migration and blocked TGFß-induced potentiation suggesting existence of a feedback loop. TGFß and Furin were also critical for hypoxia-induced BBB dysfunction. TGFß treatment aggravated hypoxia-induced BBB permeability but ALK5 or Furin blockade reversed injury-induced permeability changes. Thus during insult Furin compromises endothelial integrity by mediating the effects of TGFß. Targeting the Furin or ALK5 pathway may offer novel therapeutic strategies for improving BBB stability and CNS function during disease.

Outgrowth, proliferation, viability, angiogenesis and phenotype of primary human endothelial cells in different purchasable endothelial culture media: feed wisely.

Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.

Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent.

BACKGROUND: In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. METHODS: All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. RESULTS: The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. CONCLUSION: These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses.

Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells.

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB5 toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.

Isolation of Primary Human and Rodent Brain Microvascular Endothelial Cells: Culturing, Characterization, and High-Efficiency Transfection.

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

AIM: To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. MATERIALS & METHODS: The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. RESULTS: MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. CONCLUSION: This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

Functional behavior and gene expression of magnetic nanoparticle-loaded primary endothelial cells for targeting vascular stents.

AIM: To assess functional competence and gene expression of magnetic nanoparticle (MNP)-loaded primary endothelial cells (ECs) as potential cell-based therapy vectors. MATERIALS & METHODS: A quantitative tube formation, nitric oxide and adhesion assays were conducted to assess functional potency of the MNP-loaded ECs. A quantitative real-time PCR was used to profile genes in both MNP-loaded at static conditions and in vitro targeted ECs. RESULTS: Functional behavior of MNP-loaded and unloaded cells was comparable. MNPs induce expression of genes involved in EC growth and survival, while repress genes involved in coagulation. CONCLUSION: MNPs do not adversely affect cellular function. Gene expression indicates that targeting MNP-loaded ECs to vascular stents may potentially stimulate re-endothelialization of an implant and attenuate neointimal hyperplasia.

Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes.

The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.