Large efforts have been made to target integrin αVβ3 of endothelial cells. We have successfully developed a new class of protein (Ref to as ProAgio) by rational protein design using a stable host protein, domain 1 of cell adhesion protein CD2. ProAgio is designed to target integrin αVβ3 at a novel site and induces angiogenic endothelial cell apoptosis by recruiting and activating caspase 8 to the cytoplasmic domain of the targeted integrins. Tests with tumor xenograft models show that ProAgio strongly inhibits tumor growth. Histology analyses indicate that tumor vessels are reduced, while the established vasculatures are not affected. Toxicity analyses demonstrate that ProAgio is not toxic to mouse. Our study develops an effective anti-angiogenesis agent and provides a new platform for development of therapeutics by targeting integrins. We have successfully developed an anti-angiogenesis protein targeting integrin αVβ3 at a novel site by rational protein design. The developed agent is not toxic to non-cancerous blood vessels and other tissue/organs, providing an excellent candidate for future potential clinical development. Our developed protein is one of the very few examples that do not act through targeting VEGF/VEGFR or any other RTK pathways. The βA groove is present in almost all other β integrins. This approach may be applicable to develop agents targeting the similar βA groove of other integrin pairs, which can address wide array of pathological conditions such as AMD, Rheumatoid Arthritis, Osteoporosis etc.

Charles Univerzity in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Rebeka Klingová Supervisor: PharmDr. Petra Fikrová, Ph.D. Title of diploma thesis: Effect of soluble endoglin to endothelial cells in vitro Aim: We determined the effects of soluble endoglin on endothelial cells, by the means of inflammatory markers. The aim of the study was to point out the possible association of the soluble endoglin with endothelial dysfunction. Methods: For our study we have selected the human endothelial cells from umbilical vein - HUVEC. We have influenced the cells with soluble endoglin in two concentrations and at two different time intervals. The results were evaluated in the statistical program, in which we have compared the control group with influenced cells and two concentrations between each other. The expressions of inflammatory markers were analyzed on the level of mRNA, using the real-time PCR method. Results: Significant changes in the expression of markers were observed on the vascular and intracellular adhesion molecule at both concentrations compared to the control group. Increased values of transcription were available also for cyclooxygenase 2 and decreased values for cadherin 5 compared to the control group. Conclusion: Changes in...

Clinical manipulation of angiogenesis (the formation of new blood vessels from pre-existing vasculature) is of interest to treat diseases such as cancer and ischemic tissue where it is not properly regulated. Several treatments targeting vascular endothelial growth factor (VEGF) and its receptors - which are abundant at sites of angiogenesis - are currently in use to treat various types of cancer, however they have severe vascular side effects. Conversely, VEGF has been used clinically to promote angiogenesis to treat ischemic tissue. However, despite encouraging data from pre-clinical models, trials in humans have been disappointing. For further therapies to be developed, more information on how VEGF interacts with its receptors is required. Heparan sulfate (HS) is a ubiquitous glycosaminoglycan involved in a number of physiological processes including angiogenesis. HS facilitates the interaction of VEGF with its receptors, which is crucial for angiogenesis. Modification of this interaction via synthetic mimetics of HS may allow clinical intervention of angiogenesis. The current investigation aims first, to clarify the requirement for the interaction between VEGF and HS in angiogenesis; second to characterise the structure of HS that binds to VEGF so that mimetics can be developed; and third, to determine the effect of HS mimetics on angiogenesis in vivo. To determine the requirement for VEGF/HS interaction in angiogenesis, several mutants of VEGF165 that had lower affinities for HS were assayed for their ability to induce ectopic angiogenesis in the subintestinal baskets of zebrafish embryos. Wild type VEGF165 induced a 200-250% increase in ectopic vessels, which was matched only by a control mutant. Other mutants did not induce ectopic vessels, suggesting that this interaction is required for angiogenesis. To characterise the structure of HS that binds to VEGF, various HS mimetics were assayed against heparin in a VEGF competition assay using Biacore. Of these, the strongest inhibition (IC¬50 =~16nM) was with 2O10, an oligosaccharide that consisted of two highly sulfated octasaccharide domains (NS domains) that flanked an unsulfated dodecasaccharide region. To determine the type of sulfation required for this interaction, HS fragments were assayed for interaction with VEGF165 using the filter binding assay, and analysed by HPLC which indicated 6-O sulfation may be preferential for VEGF binding to HS.To investigate the ability of HS to affect angiogenesis, the effects of HS mimetics on zebrafish embryo subintestinal baskets were measured. The most interesting of these was with 2O10, which had a biphasic response whereby low doses (3ng) increased basket vasculature by 30% and high doses (30ng) decreased the endogenous vessels by 20%. As 2O10 had a high affinity for VEGF, its effects on the vasculature may be due to interaction with endogenous VEGF, which would indicate that HS mimetics can be used to control angiogenesis by modification of growth factor signalling. The investigation concludes that the interaction between VEGF and HS is critical for angiogenesis, and that this can be modulated by the application of HS mimetics that bind strongly to VEGF.

The differentiation of mesenchymal stem cells (MSCs) to endothelium is a critical but poorly understood feature of tissue vascularisation and considerable scepticism still remains surrounding this important differentiation event. Defining features of endothelial cells (ECs) are their ability to exist as contact-inhibited polarised monolayers that are stabilised by intercellular junctions, and the expression and activity of endothelial markers. During vasculogenesis, communication between MSCs and differentiated ECs or vascular smooth muscle cells, or between MSCs themselves is likely to influence MSC differentiation. In this study, the possibility that cell density can influence MSC differentiation along the EC lineage was examined. High density plating of human bone marrow-derived MSCs induced prominent endothelial characteristics including cobblestone-like morphology, enhanced endothelial networks, acetylated-low density lipoprotein uptake, vascular growth and stimulated expression of characteristic endothelial markers. Mechanistically, this density-dependent process has been defined. Cell-cell contact-induced Notch signalling was a key initiating step regulating commitment towards an EC lineage, whilst VEGF-A stimulation was required to consolidate the EC fate. Thus, this study not only provides evidence that MSC density is an essential microenvironmental factor stimulating the in vitro differentiation of MSCs to ECs but also demonstrates that MSCs can be differentiated to a functional EC. Taken together, defining how these crucial MSC differentiation events are regulated in vitro, provides an insight into how MSCs differentiate to ECs during postnatal neovascularisation and an opportunity for the therapeutic manipulation of MSCs in vivo, enabling targeted modulation of neovascularisation in ischaemia, wound healing and tumourigenesis.

Ageing poses one of the largest risk factors for the development of cardiovascular disease (CVD). The increased propensity towards vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells (EPCs) to contribute to vascular repair and regeneration. Among all current putative EPC populations, outgrowth endothelial cells (OECs) display the most features consistent with a human postnatal vasculogenic cell. Cell-surface heparan sulfate (HS) proteoglycans, by virtue of specific sulfated domains within the glycosaminoglycan chain, are able to bind and modulate the activities of a variety of proteins important for EPC mobilisation, homing and function at sites requiring neovascularization. This study aimed to determine if human OEC function is impaired with age, and to ascertain whether this is accompanied by changes in the fine structure of OEC HS.Using in vitro cell culture methods, OECs were isolated from healthy subjects across an age range and cell phenotype was verified by the demonstration of numerous endothelial, but not hematopoietic, cell characteristics. The functional capacity of peripheral blood derived OECs from young and old subjects, and comparative cord blood derived OECs, was assessed in terms of their susceptibility to apoptosis, proliferative, migratory and tube-forming capabilities. In vitro scratch and transwell migration assays revealed that the migratory capacity of peripheral blood derived OECs isolated from old subjects was impaired in comparison to those from young subjects and cord blood derived OECs. Structural analysis of HS by high performance liquid chromatography (HPLC) demonstrated a significant reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC HS with age (r = -0.847, p=<0.01). Moreover, a decline in the migratory response of OECs towards a gradient of VEGF significantly correlated with the percentage expression of this disaccharide (r = 0.840, p<0.01). Disruption of cell surface HS by pre-treatment with heparinase I and III was found to significantly reduce the VEGF-induced migratory response of peripheral blood derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Understanding the role of HS in regulating the directional migration of EPCs to sites requiring neovascularization and developing approaches to facilitate EPC migration may aid in the design of more successful strategies to optimise the regenerative capacity of these cells in the ageing vasculature.

There is increasing evidence that inflammatory processes play a pivotal role in the pathophysiology of ischaemic brain injury. Cerebrovascular endothelial cells that form the blood-brain barrier are critical for maintaining brain homeostasis, however, during cerebral ischaemia they contribute to the post-ischaemic inflammatory responses. It is not yet fully understood how different cerebral cells interact during this inflammatory response. This study aimed to test the hypothesis that oxygen-glucose deprivation (OGD) induces the inflammatory activation of the cerebrovascular endothelium and glial cells in vitro and that intercommunication between these cells regulate their responses to OGD. Primary murine brain endothelial cells (MBECs) monocultures, murine mixed-glial monocultures and MBEC-glial co-cultures were exposed to OGD for up to 24 hours (h), then reperfused cultures were returned to normoxia for a further 24 hours. MBECs and glia remained viable over a 24 h OGD exposure and during reperfusion. OGD induced a time-dependent increase in MBEC glucose transporter 1 (GLUT-1) expression but a time-dependent decline in expression and secretion of monocyte chemoattractant protein-1 (MCP-1). A significant increase in keratinocyte-derived chemokine (KC) secretion by MBEC monocultures was observed during reperfusion after prolonged exposure (18-24 h) to OGD whereas, KC secretion by co-cultured MBECs was increased during reperfusion after short exposure (4 h) to OGD. Co-cultured MBECs displayed a significant increase in intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in response to a short or prolonged exposure to OGD with 24 h of reperfusion. Neither OGD nor reperfusion had any effect on permeability of the MBEC monolayer. OGD induced a time-dependent increase in nuclear stabilisation of hypoxia inducible factor-1 alpha (HIF-1α) in glial cells which correlated to vascular endothelial growth factor (VEGF) secretion during OGD and subsequent reperfusion. Nuclear stabilisation of the nuclear factor kappa B (NFκB)p65 subunit by glial cells was dependent upon the duration of OGD. Reperfusion induced a significant increase in KC secretion by co-cultured glial cells after short exposure to OGD. Inflammatory activation of co-cultured MBECs and glia after 4 or 24 h OGD caused a significant increase in neutrophil transendothelial migration which correlated with MBEC expression of ICAM-1 and VCAM-1. A combination of these cell adhesion molecules with neutrophil integrins and soluble glial-derived mediators contributed to neutrophil transendothelial migration. These studies provide evidence that combined hypoxia and glucose withdrawal induces the activation of MBECs and glial cells in vitro. Cross-talk between these two cell types may further regulate their activation. As a result of this inflammatory activation, soluble MBEC and glial-derived mediators may contribute to neutrophil transendothelial migration through the regulation of MBEC cell adhesion molecule expression.

Disruption of the blood-brain barrier (BBB) is hypothesized to play an important role in the disease biology of schizophrenia (SZ). Brain microvascular endothelial cells (BMECs) have paracellular and transcellular proteins, transporters, as well as important extracellular matrix proteins, which collectively contribute to maintaining proper BBB function. While previous studies have provided some insights into the role of the BBB in SZ pathophysiology, there is a significant gap in our understanding of the cellular-molecular underpinnings of its major component, BMECs. Human induced pluripotent stem cells (hiPSCs) provide an exciting new avenue for exploring the role of BMECs in SZ. We hypothesize that BMECs have intrinsic deficits that lead to BBB dysfunction in SZ. In this study, we first aimed to test whether the existing hiPSC-derived BMEC protocols work with our patient-specific hiPSC samples. Secondly, we sought to investigate any potential deficits between BMECs derived from healthy control (HC) and SZ subjects. We successfully adapted the established protocol and confirmed the identity of these hiPSC-derived BMECs with relevant cell markers such as CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. We also evaluate barrier function by measuring trans-endothelial electrical resistance (TEER) and efflux transporters activity of ABCB1 and ABCC1. We observed evidence of poor cellular adhesion and disrupted tight junctions in a subset of SZ hiPSC-derived BMECs, where approximately 70% of them demonstrated extensive BBB disruption (reduced TEER). These findings suggest that there may be cell-autonomous disease-specific deficits in BMECs in SZ that result in BBB dysfunction. / 2022-06-07T00:00:00Z

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