Hypoxia alters biophysical properties of endothelial cells via p38 MAPK- and Rho kinase-dependent pathways

Hypoxia alters the barrier function of the endothelial cells that line the pulmonary vasculature, but underlying biophysical mechanisms remain unclear. Using rat pulmonary microvascular endothelial cells (RPMEC) in culture, we report herein changes in biophysical properties, both in space and in time, that occur in response to hypoxia. We address also the molecular basis of these changes. At the level of the single cell, we measured cell stiffness, the distribution of traction forces exerted by the cell on its substrate, and spontaneous nanoscale motions of microbeads tightly bound to the cytoskeleton (CSK). Hypoxia increased cell stiffness and traction forces by a mechanism that was dependent on the activation of Rho kinase. These changes were followed by p38-mediated decreases in spontaneous bead motions, indicating stabilization of local cellular-extracellular matrix (ECM) tethering interactions. Cells overexpressing phospho-mimicking small heat shock protein (HSP27-PM), a downstream effector of p38, exhibited decreases in spontaneous bead motions that correlated with increases in actin polymerization in these cells. Together, these findings suggest that hypoxia differentially regulates endothelial cell contraction and cellular-ECM adhesion. endothelial barrier; cytoskeleton; actin dynamics; stiffness; tensile stress     

Human lymphatic endothelial cells express multiple functional TLRs

The lymphatic endothelium is the preferred route for the drainage of interstitial fluid from tissues and also serves as a conduit for peripheral dendritic cells (DCs) to reach draining lymph nodes. Lymphatic endothelial cells (LECs) are known to produce chemokines that recruit Ag-loaded DCs to lymphatic vessels and therefore are likely to regulate the migration of DCs to lymph nodes. TLRs are immune receptors that recognize pathogen associated molecular patterns and then signal and stimulate production of inflammatory chemokines and cytokines that contribute to innate and adaptive immune responses. TLRs are known to be expressed by a wide variety of cell types including leukocytes, epithelial cells, and endothelial cells. Because the TLR expression profile of LECs remains largely unexamined, we have undertaken a comprehensive study of the expression of TLR1-10 mRNAs and protein in primary human dermal (HD) and lung LECs as well as in htert-HDLECs, which display a longer life-span than HDLECs. We found that all three cell types expressed TLR1-6 and TLR9. The responsiveness of these LECs to a panel of ligands for TLR1-9 was measured by real-time RT-PCR, ELISA, and flow cytometry, and revealed that the LECs responded to most but not all TLR ligands by increasing expression of inflammatory chemokines, cytokines, and adhesion molecules. These findings provide insight into the ability of cells of the lymphatic vasculature to respond to pathogens and potential vaccine adjuvants and shape peripheral environments in which DCs will acquire Ag and environmental cues.     

Hypoxia/Notch signaling in primary culture of rat lymphatic endothelial cells

We have developed an improved intralumenal digestion method to get a long-term primary culture of rat lymphatic endothelial cells (rLECs) that maintained their original phenotypes. rLECs in vitro under hypoxia retained their original lymphatic properties observed in the thoracic duct. Blocking Notch signal with a gamma-secretase inhibitor and transfection of a cDNA expressing a dominant negative form of Delta1 indicated that Notch signal downregulated VEGFR-2 under hypoxia and inhibited cell migration. These findings indicated that Notch signal was still operative in mature lymphatic endothelial cells in response to the oxygen concentration.     

Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells

The blood-brain barrier (BBB) serves as a critical organ in the maintenance of central nervous system homeostasis and is disrupted in a number of neurological disorders, including stroke. We examined the effects of hypoxia/aglycemia on the expression and localization of tight junction proteins, and on the function of the BBB in an in vitro model system. A receptor-operated/store-operated calcium channel blocker, SKF 96365, was used to determine if calcium flux was important in mediating hypoxia/aglycemia effects on the BBB. Expression of the tight junction protein occludin increased after hypoxic/aglycemic stress when cells were exposed to SKF 96365; this was correlated with partial protection of membrane localization of occludin and inhibition of the hypoxia-induced increase in permeability. Actin expression was dramatically reduced by hypoxia/aglycemia. Treatment with SKF 96365 during hypoxic stress protected monolayer permeability of sucrose, but transendothelial electrical resistances decreased with exposure to hypoxic stress regardless of treatment. Therefore, the presence of occludin at the membrane is dependent in part on calcium-sensitive signaling cascades; this provides a target for therapeutic intervention to minimize BBB disruption after stroke.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism

BACKGROUND: The lymphatic endothelium is an important semi-permeable barrier separating lymph from the interstitial space. However, there is currently a limited understanding of the lymphatic endothelial barrier and the mechanisms of lymph formation. The objectives of this study were to investigate the potential active role of lymphatic endothelial cells in barrier regulation, and to test whether the endothelial cell agonists VEGF-A and VEGF-C can alter lymphatic endothelial barrier function. METHODS AND RESULTS: Cultured adult human dermal microlymphatic endothelial cells (HMLEC-d) and human umbilical vein endothelial cells (HUVEC) were respectively used as models of lymphatic and vascular endothelium. Transendothelial electrical resistance (TER) of endothelial monolayers served as an index of barrier function. Cells were treated with VEGF-A, VEGF-C, or the VEGFR-3 selective mutant VEGF-C156S. MAZ51 was used to inhibit VEGFR-3 signaling. The results show that while VEGF-A causes a time-dependent decrease in TER in HUVEC, there is no response in HMLEC-d. In contrast, VEGF-C and VEGF-C156S cause a similar decrease in TER in HMLEC-d that is not observed in HUVEC. These results corresponded to the protein expression of VEGFR-2 and VEGFR-3 in these cell types, determined by Western blotting. In addition, the VEGF-C- and VEGF-C156S-induced TER changes were inhibited by MAZ51. CONCLUSIONS: The results indicate differential responses of the lymphatic and vascular endothelial barriers to VEGF-A and VEGF-C. Furthermore, our data suggest that VEGF-C alters lymphatic endothelial function through a mechanism involving VEGFR-3.     

Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering

In order to provide a suitable source of cells for lymphatic tissue engineering, the present study was designed to investigate techniques for harvesting and cryopreservation of human dermal lymphatic endothelial cells (LECs) in vitro. The LECs were isolated from children’s foreskins and then cultured in endothelial growth medium-2 MV (EGM-2-MV) with 5% FBS. The second passage LECs were suspended in cryopreservation solution containing 40% FBS and 10% Me₂SO in EGM-2-MV, cooled to −80 °C at about 1 °C/min and stored in liquid nitrogen. Samples were thawed quickly in a 37 °C water bath, and the cryoprotectant was removed by serial elution. The membrane integrity of thawed LECs was determined by trypan blue staining exclusion, and their proliferation was evaluated using the MTT method. The expanded cells of two groups were identified using immunofluorescence staining and RT-PCR with lymphatic-specific markers such as Podoplanin and VEGFR-3. Uptake of fluorescent DiI-Ac-LDL and microtubular formation in three-dimensional cultures were used to detect the function of LECs. Flow cytometry was applied to identify cells and to measure the apoptosis rate as well. Cryopreservation resulted in a retrieval of 67 ± 4% and an intact cell rate of 80 ± 3%. The early apoptosis rate of thawed LECs (9.15 ± 0.34%) was higher than that of fresh control LECs (5.31 ± 0.23%). The growth curves of thawed LECs were similar to those of fresh LECs. The thawed LECs were propagated for at least 6-7 passages without alterations in phenotype and function. Highly purified LECs can be isolated by immunomagnetic beads from human dermis. The cryopreserved/thawed and recultivated LECs are proven to have high vitality and growth potential in vitro and may be considered suitable seed cells for lymphatic tissue engineering.     

Hypoxia differentially regulates VEGFR1 and VEGFR2 levels and alters intracellular signaling and cell migration in endothelial cells

The role of hypoxia on endothelial cell function and response to growth factors is unknown. Here, we tested the hypothesis that hypoxia re-programs endothelial function by modulating vascular endothelial growth factor receptor levels which in turn alter intracellular signaling and cell function. Hypoxia stimulated VEGF-A and VEGFR1 expression but decreased VEGFR2 levels in endothelial cells. During hypoxia, plasma membrane VEGFR1 levels were elevated whereas VEGFR2 levels were depleted. One functional consequence of hypoxia is a reduction in VEGF-A-stimulated and VEGFR2-regulated intracellular signaling including lowered endothelial nitric oxide synthase activation. Venous, arterial and capillary endothelial cells subjected to hypoxia all exhibited reduced cell migration in response to VEGF-A. A mechanistic explanation is that VEGFR1:VEGFR2 ratio is substantially increased during hypoxia to block VEGF-A-stimulated and VEGFR2-regulated endothelial responses to maximize cell viability and recovery.     

Gene expression profile of lymphatic endothelial cells

The lymphatic system was first described at around the same time as the blood circulation centuries ago, but the biological function elucidation of LECs (lymphatic endothelial cells) is far less than that of BVECs (blood vascular endothelial cells). Since the discovery of molecular markers for LECs and exploration of lymphatic role in tumour metastasis, more attention has been given to basic lymphatic research. Approx. 150 known genes were found to be expressed at the mRNA and protein levels by LECs. These molecules play an important role in lymphangiogenesis, signalling, tumour metastasis, immune function and fluid transport. This review provides a brief outline of gene expression profile of LECs and the molecular biological function, which will give the reader a better understanding about the mechanics of lymphatic function and some pathologies related to the lymphatic system such as lymphoedema, and facilitate advanced scientific research into lymphatic biology.     

Distinct adhesive properties of granulocytes and monocytes to endothelial cells under static and stirred conditions

Adherence of neutrophils and monocytes to endothelium is an important event in the sequence of leukocyte responses to inflammatory disease. Double-color flow microfluorimetry analysis was used to determine neutrophil and monocyte adherence to suspended endothelial cells under stirred conditions. The static adherence to endothelial cell monolayers was simultaneously determined. In both assays, neutrophils behaved in a similar way. Basal adherence of neutrophils was very low. Activation by PMA or by the chemoattractants platelet-activating factor and FMLP induced rapid binding, primarily mediated by CR3. Nonactivated neutrophils showed CD18-dependent (lymphocyte function-associated Ag-1 and CR3) and CD18-independent adherence to endothelial cells when the endothelial cells were prestimulated with rIL-1 beta. In contrast to neutrophils, nonactivated monocytes adhered avidly to resting endothelial cells. This adherence was partly CD18 dependent and partly CD18 independent. Whereas monocyte adherence under stirred conditions strongly increased upon activation by PMA, a significant decrease in adherence was found under static conditions, which was prevented by cytochalasin B. This decrease was limited to a distinct CD18-independent binding mechanism, and absent under stirred conditions. We conclude that monocytes adhere with (at least) three binding mechanisms to endothelial cells, a CD18-dependent and two CD18-independent mechanisms.     

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.     

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).     

Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury.

The effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time-dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate-treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia-enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge.     

Characteristics of lymphatic endothelial cells in physiological and pathological conditions

Impairment of lymphatic structure and function, e.g., inadequate endothelial permeability and intercellular openings, abnormal lymphangiogenesis and overexpression for immunoreactive agents, will result in tumor metastasis, autoimmune response alteration and accumulation of interstitial fluid and proteins. Recently, several novel molecules have been identified that allow a more precise distinction between lymphatic and blood vascular endothelium. The differences in expression of endothelial markers on the lymphatic vessel strongly suggest the possibility that there will be important divergence in the differentiating and regenerating responses in lymphatic behavior to various pathological processes. Undoubtfully, molecular techniques would also lead to the definition of unique markers found on lymphatic endothelial cells (LECs) in lymphatic-associated diseases which are mostly involved in lymphangiogenesis. This review is mainly concentrated on the characteristics of LECs in diabetes, wound healing, lymphedema and tumor, especially in the experimental models that have offered insight into the LEC role in these diseases affecting the lymphatic system. Increased knowledge of the molecular signaling pathways driving lymphatic development and lymphangiogenesis should boost the impact of therapeutics on the diseases. Although the field about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels has experienced rapid progress in the past few years, an understanding of the basis of the differences and their implications in the pathological conditions will require much more investigation.