Endothelial Cell-cell Interactions Research Articles

Endothelial activation, Cell-Cell Interactions, and Inflammatory Pathways in Postoperative Atrial Fibrillation Following Cardiac Surgery.

Postoperative atrial fibrillation (POAF) is common after cardiac surgery and related to endothelial activation and systemic inflammation. Herein, we investigate the pathophysiological mechanisms of AF through endothelial activation and cell-cell interactions related to the development of POAF. Patients without previous AF undergoing cardiac surgery were studied. Permanent AF patients were included as positive controls. Interleukin (IL)-6, Von Willebrand factor (vWF), N-terminal pro-brain natriuretic peptide (NT-proBNP) and high sensitivity troponin T (hsTnT) were evaluated by electrochemiluminescence. Vascular cell adhesion molecule-1 (VCAM-1) and human Growth Differentiation Factor 15 (GDF-15) was assessed by ELISA. Connexins (Cxs) 40 and 43 were measured by tissue immunolabelling, and apoptosis by TUNEL assay. We included 117 patients (median age 67: 27.8% female): 17 with permanent AF; 27 with POAF and 73 with non- AF. Patients with permanent AF and POAF had higher levels of NT-proBNP, hs-TnT, apoptotic nuclei and decrease Cx43 expression, compared to non-AF patients (all p-value <0.05). VCAM-1 and GDF-15 were significantly higher in permanent AF vs. non-AF (p=0.013 and p=0.035). Greater endothelial activation and inflammation in AF patients compared to those without AF was found. The proinflammatory state in AF patients, in addition to the lower expression of Cx43, seems to be associated with atrial remodeling processes occurring in AF.

Oscillatory contractile forces refine endothelial cell-cell interactions for continuous lumen formation governed by Heg1/Ccm1

The formation and organization of complex blood vessel networks rely on various biophysical forces, yet the mechanisms governing endothelial cell-cell interactions under different mechanical inputs are not well understood. Using the dorsal longitudinal anastomotic vessel (DLAV) in zebrafish as a model, we studied the roles of multiple biophysical inputs and cerebral cavernous malformation (CCM)-related genes in angiogenesis. Our research identifies heg1 and krit1 (ccm1) as crucial for the formation of endothelial cell-cell interfaces during anastomosis. In mutants of these genes, cell-cell interfaces are entangled with fragmented apical domains. A Heg1 live reporter demonstrated that Heg1 is dynamically involved in the oscillatory constrictions along cell-cell junctions, whilst a Myosin live reporter indicated that heg1 and krit1 mutants lack actomyosin contractility along these junctions. In wild-type embryos, the oscillatory contractile forces at junctions refine endothelial cell-cell interactions by straightening junctions and eliminating excessive cell-cell interfaces. Conversely, in the absence of junctional contractility, the cell-cell interfaces become entangled and prone to collapse in both mutants, preventing the formation of a continuous luminal space. By restoring junctional contractility via optogenetic activation of RhoA, contorted junctions are straightened and disentangled. Additionally, haemodynamic forces complement actomyosin contractile forces in resolving entangled cell-cell interfaces in both wild-type and mutant embryos. Overall, our study reveals that oscillatory contractile forces governed by Heg1 and Krit1 are essential for maintaining proper endothelial cell-cell interfaces and thus for the formation of a continuous luminal space, which is essential to generate a functional vasculature.

Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells.

The integrity of retinal endothelial cell (EC) is essential for establishing and maintaining the retinal blood barrier to ensure proper vision. Vitamin D is a hormone with known protective roles in EC function. The majority of vitamin D action is mediated through the vitamin D receptor (VDR). VDR is a nuclear receptor whose engagement by vitamin D impacts the expression of many genes with important roles in regulation of angiogenesis and inflammation. Although many studies have investigated vitamin D-VDR action in cardiovascular protection and tumor angiogenesis, its impact on retinal EC function and regulation of ocular angiogenesis and inflammation is exceedingly limited. We previously showed calcitriol, the active form of vitamin D, is a potent inhibitor of retinal neovascularization in vivo and retinal EC capillary morphogenesis in vitro. Here, using retinal EC prepared from wild-type (Vdr+/+) and VDR-deficient (Vdr-/-) mice, we show that retinal EC express VDR and its expression is induced by calcitriol. The lack of VDR expression had a significant impact on endothelial cell-cell and cell-matrix interactions. Vdr-/- retinal EC proliferated at a slower rate and were more adherent and less migratory. They also exhibited increased expression levels of inflammatory markers driven in part by sustained activation of STAT1 and NF-κB pathways and were more sensitive to oxidative challenge. These changes were attributed, in part, to down-regulation of endothelial nitric oxide synthetase, enhanced hepcidin expression, and increased intracellular iron levels. Taken together, our results indicate that VDR expression plays a fundamental role in maintaining the proper angiogenic and inflammatory state of retinal EC.

Abstract 5976: Engineering easy-to-implement [18F] nanobody-PET for emerging immuno-onco target, ICAM-1

Abstract Intercellular adhesion molecule-1 (ICAM-1) participates in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability. ICAM-1 is highly expressed in inflammatory conditions, chronic diseases and several cancers, like anaplastic thyroid cancer (ATC) and triple negative breast cancer (TNBC). This rationale has motivated the pursuit of ICAM-1 as an emerging Immuno-Onco target, with many groups developing adoptive cell therapies against ICAM-1. Visualizing the ICAM-1 expression in the patients noninvasively is critical for a successful treatment. Identifying the ideal candidate patient, track the therapeutic effect of these treatments, the changes implicated in these diseases, or even after therapy for efficacy. PET imaging would be ideal due to its quantifiability, but unfortunately so far there is no such PET tracer to image ICAM-1 in patients. Here, we developed a nanobody based PET platform to image ICAM-1. Nanobodies are the smallest known functional antibody fragment. They are derived from heavy-chain only antibodies (HcAbs) in camelids. They retain high affinity and specificity for their target antigens, with low off-target accumulation due to their hydrophilic properties. Their nanoscale dimensions enable deep penetration of tumors, tolerance of high temperatures, elevated pressures, non-physiological pH, and even strong chemical denaturants. We generated these nanobodies by immunizing alpaca with Fc-fused human ICAM-1, produced and purified from mammalian cell transfection media. The harvested B cells from these immunized alpaca were used to generate a cDNA library, containing transcripts of the VHH domain of the HcAbs. This cDNA library was subsequently cloned into a yeast-surface display platform. After three rounds of panning we screened 160+ ICAM-1 binding clones from which we identified 8 unique clones with high to moderate binding to ICAM-1. Among which, clone A7 was utilized for nanobody-PET due to its high affinity (~115nM) and specificity to ICAM-1. With the goal of having a nanobody-PET that would be easy to execute at a moment’s notice and translated into clinic, we decided to utilize NOTA/aluminum-fluoride chemistry to radio label our PET tracer. For consistency in labeling, we utilized sortase to label the nanobody with NOTA at a 1:1 ratio. We demonstrated the utility of this ICAM-1 nanobody-PET tracer in subcutaneous tumor mouse xenograft model. PET/CT imaging shows clear localization to ICAM-1-positive xenograft, with low background uptake apart from minor gallbladder and kidney clearance. The success of this tracer in conjunction with the ease of NOTA/aluminum-fluoride chemistry opens up the possibility to carry over nanobody-PET to other targets (e.g., MSLN, EpCAM). Citation Format: Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Yago Alcaina, Yanping Yang, Moonsoo M. Jin. Engineering easy-to-implement [18F] nanobody-PET for emerging immuno-onco target, ICAM-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5976.

BIOL-01. THE RELATIONSHIP OF BRAIN ENDOTHELIAL WNT SIGNAL INHIBITION ON BLOOD-TUMOR BARRIER INTEGRITY

The blood-tumor barrier (BTB) is the primary site of nutrient and drug transport to tumor cells such as malignant gliomas. Yet, signaling pathways and factors influencing BTB permeability are poorly understood. Previous studies demonstrate the role of WNT/β-catenin signaling in establishing and fortifying blood-brain barrier integrity in a non-diseased state. Additionally, WNT proteins are highly expressed in gliomas and their surrounding vasculature. Thus, we propose inhibition of WNT/β-catenin signaling at the brain endothelium of malignant glioma can impair BTB integrity to enhance permeability for select cytotoxic agents. We used immortalized mouse brain endothelial cells (bEnd.3), akin to brain tumor endothelium, treated for 24 hours with WNT inhibitors (ICG-001, IWR-1, and LGK974). Inhibition of WNT/β-catenin signaling was confirmed by gene expression of transcription factors (Tcf4 and Birc5). Cell viability was confirmed by CellTiter Glo®. Brain endothelial cell-cell interaction was evaluated by cell impedance and resistance via the Agilent xCELLigence and ABP TEER24 systems. Using qPCR and flow cytometry, we observed changes in expression and function of Abcb1 and Abcg2 transporters. Using an in vitro BTB (bEnd.3 cells and mouse H3.3WT/K27 glioma cells) we evaluated the effect of WNT inhibition on permeability and glioma viability. We found that all the inhibitors downregulated Tcf4 and Birc5 in brain endothelium dose-dependently. Viability with inhibitors demonstrated an IC50 of 28μM for ICG-001, and 42μM for both IWR-1 and LGK974. Endothelial cell-cell interaction was transiently decreased by approximately 50% with all inhibitors at 30 minutes; increasing closer to baseline after 2-4hrs. All WNT inhibitors dose-dependently decreased Abcg2 transporter expression and function. While In vitro BTB studies are ongoing, preliminary findings demonstrate increasing permeability of BTB amongst H3.3K27 glioma cells. Our results demonstrate potential of WNT inhibitors to modulate BTB integrity and drug efflux function. More studies are warranted to explore WNT/β-catenin signaling inhibition on BTB in vivo.

SCIDOT-21. IMPROVING DRUG DELIVERY TO GLIOBLASTOMA BY TARGETING CANONICAL WNT/β-CATENIN SIGNALING IN THE BLOOD-BRAIN BARRIER

Abstract BACKGROUND Glioblastoma (GBM) patient survival and therapy response is greatly hindered by the presence of invasive glioma stem cells (GSC) and the blood-brain barrier (BBB) which limits effective drug delivery. WNT/beta-catenin signaling is important in the development and maintenance of the BBB by mediating transcription of growth factors, multidrug resistance proteins, and junctional proteins. In WNT-subtype medulloblastoma, activating mutations of beta-catenin lead to reciprocal secretion of WNT antagonists such as WIF1 and DKK1 into the tumor microenvironment. These WNT antagonists can act upon the surrounding endothelium and induce a leaky BBB. Therefore, we hypothesize that pharmacological inhibition of WNT/beta-catenin signaling in brain endothelial cells will decrease BBB integrity, enabling enhanced paracellular drug delivery to infiltrative GSCs. METHODS We recapitulated the WNT-medulloblastoma phenotype in GBM by activating WNT/beta-catenin signaling in primary human GSCs, inducing secretion of downstream WNT antagonists. Conditioned-media (CM) from GSCs was then applied to human brain microvascular endothelial cells (HBMEC) to indirectly inhibit WNT signaling. Additionally, we directly inhibited WNT/beta-catenin signaling in HBMECs with the small molecule inhibitor ICG-001. Endothelial cell-cell interaction was measured by electrical impedance using the ACEA xCELLigence system. Fenestration and junctional expression were evaluated by immunoblotting and immunofluorescence. RESULTS ICG-001 or WNT-GSC-CM, but not control GSC-CM, upregulated fenestration related protein, PLVAP, and downregulated junctional proteins claudin-5, ZO-1, and VE-Cadherin in HBMECs. Endothelial cell-cell interaction was transiently decreased by ICG-001 or WNT-GSC-CM. Pre-clinical studies are underway to evaluate the functional impact of WNT/beta-catenin inhibition on BBB integrity and permeability in rodent glioma models. Altogether, these results support targeting WNT/beta-catenin signaling in brain endothelial cells to enhance drug delivery to CNS tumors. CONCLUSION Modulation of intratumoral Wnt/beta-catenin signaling, particularly in highly resistant GSCs, may enhance chemotherapy drug delivery, potentially expanding the drug portfolio and improving the prognosis of GBM.

Stabilization of cell-cell junctions by active vitamin D ameliorates uraemia-induced loss of human endothelial barrier function.

Uraemia induces endothelial cell (EC) injury and impaired repair capacity, for which the underlying mechanism remains unclear. Active vitamin D (VD) may promote endothelial repair, however, the mechanism that mediates the effects of VD in chronic kidney disease are poorly understood. Thus, we investigated uraemia-induced endothelial damage and the protection against such damage by active VD. We applied electric cell-substrate impedance sensing (ECIS) to study real-time responses of human ECs exposed to pooled uraemic and non-uraemic plasma with or without the addition of active VD. The effects of indoxyl sulphate and p-cresol were tested in non-uraemic plasma. Structural changes for vascular endothelial (VE)-cadherin and F-actin were assessed by immunostaining and quantified. The exposure of ECs to uraemic media significantly decreased endothelial barrier function after 24 h. Cell migration after electrical wounding and recovery of the barrier after thrombin-induced loss of integrity were significantly impaired in uraemic-medium stimulated cells and cells exposed to indoxyl sulphate and p-cresol. This effect on ECIS was dependent on loss of cell-cell interaction. Mechanistically, we found that EC, exposed to uraemic media, displayed disrupted VE-cadherin interactions and F-actin reorganization. VD supplementation rescued both endothelial barrier function and cell-cell interactions in ECs exposed to uraemic media. These events were associated with an increment of VE-cadherin at intercellular junctions. Our data demonstrate a potentially clinically relevant mechanism for uraemia-induced endothelial damage. Furthermore, active VD rescued the uraemic medium-induced loss of cell-cell adhesion, revealing a novel role of active VD in preservation of endothelial integrity during uraemia.

The Role of Sialylated Glycans in Human Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1)-mediated Trans Homophilic Interactions and Endothelial Cell Barrier Function

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) is a major component of the endothelial cell intercellular junction. Previous studies have shown that PECAM-1 homophilic interactions, mediated by amino-terminal immunoglobulin homology domain 1, contribute to maintenance of the vascular permeability barrier and to its re-establishment following inflammatory or thrombotic insult. PECAM-1 glycans account for ∼30% of its molecular mass, and the newly solved crystal structure of human PECAM-1 immunoglobulin homology domain 1 reveals that a glycan emanating from the asparagine residue at position 25 (Asn-25) is located within the trans homophilic-binding interface, suggesting a role for an Asn-25-associated glycan in PECAM-1 homophilic interactions. In support of this possibility, unbiased molecular docking studies revealed that negatively charged α2,3 sialic acid moieties bind tightly to a groove within the PECAM-1 homophilic interface in an orientation that favors the formation of an electrostatic bridge with positively charged Lys-89, mutation of which has been shown previously to disrupt PECAM-1-mediated homophilic binding. To verify the contribution of the Asn-25 glycan to endothelial barrier function, we generated an N25Q mutant form of PECAM-1 that is not glycosylated at this position and examined its ability to contribute to vascular integrity in endothelial cell-like REN cells. Confocal microscopy showed that although N25Q PECAM-1 concentrates normally at cell-cell junctions, the ability of this mutant form of PECAM-1 to support re-establishment of a permeability barrier following disruption with thrombin was significantly compromised. Taken together, these data suggest that a sialic acid-containing glycan emanating from Asn-25 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface.

HIF-2α-mediated induction of pulmonary thrombospondin-1 contributes to hypoxia-driven vascular remodelling and vasoconstriction.

AimsHypoxic conditions stimulate pulmonary vasoconstriction and vascular remodelling, both pathognomonic changes in pulmonary arterial hypertension (PAH). The secreted protein thrombospondin-1 (TSP1) is involved in the maintenance of lung homeostasis. New work identified a role for TSP1 in promoting PAH. Nonetheless, it is largely unknown how hypoxia regulates TSP1 in the lung and whether this contributes to pathological events during PAH.Methods and resultsIn cell and animal experiments, we found that hypoxia induces TSP1 in lungs, pulmonary artery smooth muscle cells and endothelial cells, and pulmonary fibroblasts. Using a murine model of constitutive hypoxia, gene silencing, and luciferase reporter experiments, we found that hypoxia-mediated induction of pulmonary TSP1 is a hypoxia-inducible factor (HIF)-2α-dependent process. Additionally, hypoxic tsp1−/− pulmonary fibroblasts and pulmonary artery smooth muscle cell displayed decreased migration compared with wild-type (WT) cells. Furthermore, hypoxia-mediated induction of TSP1 destabilized endothelial cell–cell interactions. This provides genetic evidence that TSP1 contributes to vascular remodelling during PAH. Expanding cell data to whole tissues, we found that, under hypoxia, pulmonary arteries (PAs) from WT mice had significantly decreased sensitivity to acetylcholine (Ach)-stimulated endothelial-dependent vasodilation. In contrast, hypoxic tsp1−/− PAs retained sensitivity to Ach, mediated in part by TSP1 regulation of pulmonary Kv channels. Translating these preclinical studies, we find in the lungs from individuals with end-stage PAH, both TSP1 and HIF-2α protein expression increased in the pulmonary vasculature compared with non-PAH controls.ConclusionsThese findings demonstrate that HIF-2α is clearly implicated in the TSP1 pulmonary regulation and provide new insights on its contribution to PAH-driven vascular remodelling and vasoconstriction.

Domains I and IV of Annexin A2 Affect the Formation and Integrity of In Vitro Capillary-Like Networks

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

Getting the “Inside” Scoop on EphrinB2 Signaling in Pericytes and the Effect on Peritubular Capillary Stability

Progressive CKD develops from a lack of fidelity in tissue remodeling after acute injury. A lack of successful repair following even subtle injuries, regardless of their cause, suggests that complete resolution of function may be difficult to achieve, even if initiating deficits are corrected. This

Relocalization of Junctional Adhesion Molecule A during Inflammatory Stimulation of Brain Endothelial Cells

Junctional adhesion molecule A (JAM-A) is a unique tight junction (TJ) transmembrane protein that under basal conditions maintains endothelial cell-cell interactions but under inflammatory conditions acts as a leukocyte adhesion molecule. This study investigates the fate of JAM-A during inflammatory TJ complex remodeling and paracellular route formation in brain endothelial cells. The chemokine (C-C motif) ligand 2 (CCL2) induced JAM-A redistribution from the interendothelial cell area to the apical surface, where JAM-A played a role as a leukocyte adhesion molecule participating in transendothelial cell migration of neutrophils and monocytes. JAM-A redistribution was associated with internalization via macropinocytosis during paracellular route opening. A tracer study with dextran-Texas Red indicated that internalization occurred within a short time period (~10 min) by dextran-positive vesicles and then became sorted to dextran-positive/Rab34-positive/Rab5-positive vesicles and then Rab4-positive endosomes. By ~20 min, most internalized JAM-A moved to the brain endothelial cell apical membrane. Treatment with a macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, or Rab5/Rab4 depletion with small interfering RNA oligonucleotides prevented JAM-A relocalization, suggesting that macropinocytosis and recycling to the membrane surface occur during JAM-A redistribution. Analysis of the signaling pathways indicated involvement of RhoA and Rho kinase in JAM-A relocalization. These data provide new insights into the molecular and cellular mechanisms involved in blood-brain barrier remodeling during inflammation.

Dynamic regulation of endothelial cell-cell interactions

Dynamic regulation of endothelial cell-cell interactions

PECAM-1 regulates proangiogenic properties of endothelial cells through modulation of cell-cell and cell-matrix interactions

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a member of the immunoglobulin superfamily of cell adhesion molecules with important roles in angiogenesis and inflammation. However, the molecular and cellular mechanisms, and the role that specific PECAM-1 isoforms play in these processes, remain elusive. We recently showed attenuation of retinal vascular development and neovascularization in PECAM-1-deficient (PECAM-1-/-) mice. To gain further insight into the role of PECAM-1 in these processes, we isolated primary retinal endothelial cells (EC) from wild-type (PECAM-1+/+) and PECAM-1-/- mice. Lack of PECAM-1 had a significant impact on endothelial cell-cell and cell-matrix interactions, resulting in attenuation of cell migration and capillary morphogenesis. Mechanistically these changes were associated with a significant decrease in expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) bioavailability in PECAM-1-/- retinal EC. PECAM-1-/- retinal EC also exhibited a lower rate of apoptosis under basal and challenged conditions, consistent with their increased growth rate. Furthermore, reexpression of PECAM-1 was sufficient to restore migration and capillary morphogenesis of null cells in an isoform-specific manner. Thus PECAM-1 expression modulates proangiogenic properties of EC, and these activities are significantly influenced by alternative splicing of its cytoplasmic domain.

Myeloablative Therapies Damage and Alter the Composition of the Cells of the Bone Marrow Microenvironment.

Abstract 1447Poster Board I-470Patients with a range of blood cell diseases are usually treated with myeloablative therapies such as chemotherapy or irradiation accompanied by bone marrow (BM) transplants. These therapies have severe effects on the hematopoietic system and cause prolonged pancytopenia, which is life-threatening to the patient. The effects of myeloablative therapies on hematopoietic cells are well-documented but their impacts on the cells of the BM microenvironment, which have important roles in regulating hematopoiesis, are largely unknown. Using the mouse model, we have analyzed the effects of 11 Gy irradiation and transplantation of 5 × 106 BM cells/mouse on hematopoietic and BM microenvironment cells: osteoblasts (Obs), endothelial cells (ECs) (both which have been shown to be important positive regulators of hematopoiesis) and adipocytes (known negative regulators of hematopoiesis). Mature and immature hematopoietic cell contents in BM, peripheral blood and spleen were assessed by cell counts together with fluorescence-activated cell-sorting (FACS) analysis and colony-forming cell potential. BM microenvironment Ob and adipocyte cell content were examined by standard histomorphometry methods on non-decalcified plastic-embedded tibiae. BM microenvironment ECs were measured by immunohistochemical staining of paraffin-embedded sections accompanied by FACS-analysis of lineage/CD45/CD48-negative, CD31-positive cells. Quantitative real-time PCR of BM provided further insight into changes in transcripts specific for immature populations of BM microenvironment cells post-transplant. Cohorts of male mice were analyzed at numerous time-points corresponding to: age-matched non-transplanted male mice (day 0); periods of severe pancytopenia (days 2-7); early recovery of hematopoiesis (days 10-35) and the time-point at which stable hematopoiesis was re-established (day 84). The earliest change in the BM microenvironment was to ECs, as massive hemorrhaging was observed, with damaged vasculature throughout the BM cavity occurring as early as day 2 post-transplant. Furthermore, the numbers of ECs in the BM markedly dropped at this time-point, and this was accompanied by 2-fold reductions in transcripts for the BM vascular-specific markers VEGFR2 and VEGFR3. In contrast, the expression of VE-cadherin, a molecule important for endothelial cell-cell interactions, was significantly up-regulated (6-fold, P<0.05 vs day 0), potentially to aid repair of the vasculature. At the same time-point stromal cell-derived factor-1 (CXCL12), a chemokine that retains hematopoietic cells in the BM but also functions as an angiogenic factor, was highly up-regulated in BM (30-fold, P<0.001) but returned to normal levels by day 7 post-transplant. Interestingly, EC cytopenia paralleled that observed for hematopoietic cells, however, full recovery of BM ECs was detected by day 14 post-transplant. There was a significant 2-fold reduction in trabecular bone that persisted from days 7-35 post-transplant (P<0.05 vs day 0). This correlated with significant (2-fold) decreases in transcripts of Runx2 and Osterix (P<0.005 vs day 0), both markers of immature Ob precursors, Furthermore, while non-treated age-matched mice did not have significant numbers of adipocytes in their BM, there was a 20-fold increase in adipocytes visible from days 7-35 post-transplant (P<0.05). This was preceded by a significant increase in expression of the early adipocyte markers peroxisome proliferator-activated receptor-γ (PPARγ) (12-fold) and adiponectin (3-fold) in the BM by day 2 post-transplant (P<0.0005 vs day 0). These inverse changes observed in bone and adipocyte formation may help to explain why both parathyroid hormone and a PPARγ antagonist (which target mesenchymal progenitor cells to reduce adipogenesis and increase bone formation) have recently been shown to aid hematological recovery post-transplant. Preliminary results suggest that similar BM microenvironment cell changes are also observed when mice are treated with the cytotoxic drug 5-fluorouracil. In conclusion, the cells of the BM microenvironment are highly susceptible to myeloablative therapies. Treatments aiding the recovery of the different BM microenvironment cell types might in turn reduce pancytopenia and promote more rapid hematological recovery in patients receiving myeloablative therapies. DisclosuresNo relevant conflicts of interest to declare.

PECAM-1 isoform-specific regulation of kidney endothelial cell migration and capillary morphogenesis

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in angiogenesis through its involvement in endothelial cell-cell and cell-matrix interactions and signal transduction. Recent studies indicate that the cytoplasmic domain of PECAM-1 plays an important role in its cell adhesive and signaling properties. However, the role PECAM-1 isoforms play during angiogenic events such as cell adhesion and migration requires further delineation. To gain insight into the role PECAM-1 plays during vascular development and angiogenesis, we examined the expression pattern of PECAM-1 isoforms during kidney vascularization. We show that multiple isoforms of PECAM-1 are expressed during renal vascular development with different frequencies. The PECAM-1 that lacks exons 14 and 15 (Delta14&15) was the predominant isoform detected in the renal vasculature. To further study PECAM-1 isoform-specific functions we isolated kidney endothelial cells (EC) from wild-type and PECAM-1-deficient (PECAM-1-/-) mice with B(4)-lectin-coated magnetic beads. PECAM-1-/- kidney EC showed reduced migration, inability to undergo capillary morphogenesis in Matrigel, dense peripheral focal adhesions, and peripheral cortical actin distribution compared with wild-type cells. PECAM-1-/- kidney EC secreted increased amounts of fibronectin and decreased amounts of tenascin-C and thrombospondin-1. Reexpression of Delta14&15, but not full-length, PECAM-1 in PECAM-1-/- kidney EC restored cell migration and capillary morphogenesis defects. Thus PECAM-1 may regulate the adhesive and migratory properties of kidney EC in an isoform-specific fashion through modulation of integrin activity and extracellular matrix protein expression. Our results indicate that regulated expression of specific PECAM-1 isoforms may enable EC to accommodate the different stages of angiogenesis.

Dual Roles of Tight Junction-associated Protein, Zonula Occludens-1, in Sphingosine 1-Phosphate-mediated Endothelial Chemotaxis and Barrier Integrity

In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G(i)/Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G(i)/Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and alpha-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/alpha-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.

Probing Intercellular Interactions between Vascular Endothelial Cadherin Pairs at Single-molecule Resolution and in Living Cells

Vascular endothelial (VE) cadherin is the surface glycoprotein cadherin specific to the endothelium that mediates cell-cell adhesion and plays a major role in the remodeling, gating, and maturation of vascular vessels. To investigate the contribution of individual VE-cadherins to endothelial cell-cell interactions and investigate whether different classical cadherins display different kinetics and micromechanical properties, we characterize the binding properties of VE-cadherin/VE-cadherin bonds at single-molecule resolution and in living human umbilical vein endothelial cells (HUVECs). Our single-molecule force spectroscopy measurements reveal that type II VE-cadherin molecules form bonds that are less prone to rupture and display a higher tensile strength than bonds formed by classical type I neuronal (N) cadherin and epithelial (E) cadherin. The equilibrium lifetime of the VE-cadherin/VE-cadherin bond is significantly longer than formed by N-cadherin/N-cadherin bonds and E-cadherin/E-cadherin bonds. These results indicate that VE-cadherins form bonds that have kinetics and mechanical properties that are significantly different from those formed by classical type I cadherins, properties that are particularly well adapted to the barrier and adhesive functions of VE-cadherin in endothelial cell-cell junctions.

Receptor Protein Tyrosine Phosphatase μ Regulates the Paracellular Pathway in Human Lung Microvascular Endothelia

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

Oxidative stress-elicited myocardial apoptosis during reperfusion.

Myocardial apoptosis primarily triggered during reperfusion has been associated with cardiac dysfunction and extension of infarction. Although potential signaling pathways involved in triggering apoptosis remain to be clearly defined, an increasing number of experiments have implicated the generation of reactive oxygen species (ROS). Increased levels of ROS have been shown to cause inflammatory and endothelial cell-cell interactions and calcium overload, resulting in enhanced release of pro-apoptotic genes from mitochondria. ROS also activate mitogen-activated protein kinases, stimulate nuclear factor-kappaB and promote synthesis of tumour necrosis factor-alpha. Attenuation of apoptosis by antioxidants has opened a new therapeutic window in the treatment of ischemia/reperfusion injury.