Endothelial Cell Receptor Research Articles

Immunotherapy engagement in pancreatic adenocarcinoma: Provisional results of iLSTA study— Durvalumab, LSTA1 (certepetide), gemcitabine, and nab-paclitaxel for locally advanced pancreatic ductal adenocarcinoma.

746 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterised by a dense, extracellular matrix-rich stroma, which creates a physical barrier against drug penetration. Evidence suggests that the iRGD peptide, LSTA-1, can increase the penetration of anti-cancer drugs to tumours through selective targeting of tumour endothelial cell receptors. Additionally, LSTA-1 administration promotes CD8+ immune cell tumour infiltration, potentially enabling immunotherapy as a treatment modality. iLSTA examined safety, tolerability and effect of the combination. Methods: Participants diagnosed with locally advanced PDAC were divided into 3 cohorts in a 1:1:4 ratio. Cohort 1 (n=5) received gemcitabine (1000mg/m 2 ), nab-paclitaxel (125mg/m 2 ), placebo LSTA-1 (3.2mg/kg) and placebo durvalumab (750mg). Cohort 2 (n=5) received gemcitabine, nab-paclitaxel, active and placebo LSTA-1, and placebo durvalumab. Cohort 3 (n=20) received gemcitabine, nab-paclitaxel, LSTA-1 and durvalumab. Tissue biopsies via endoscopic ultrasound were obtained pre-treatment and between weeks 12 and 16 for analysis of tumour infiltrating lymphocytes (TILs). Patient tumour sizes were assessed every 8 weeks using radiological imaging according to RECIST v1.1, and serum CA19-9 levels were analysed monthly. Results: 6/17 patients showed significant RECIST partial response after 2 cycles of treatment (5 patients in cohort 3), with the remaining 11 patients exhibiting stable disease. After 4 cycles of treatment, 10/17 patients demonstrated partial response (9 patients in cohort 3). Of the remaining 7 patients, 6 demonstrated stable disease, and 1 patient (cohort 2) exhibited a RECIST complete response. 13/17 patients who completed 4 treatment cycles showed a decrease in CA19-9 levels. 6 patients demonstrated >90% reduction in CA19-9 (5 in cohort 3), with the remaining 7 patients showing a >50% reduction in CA19-9 levels (5 in cohort 3). 12 patients underwent repeat biopsies 12-16 weeks post-treatment to assess TILs. 10 patients showed immune cell infiltration (5% to 50% stroma infiltration), 2 had no tumour found (Cohort 3). The combination was safe with no unexpected toxicities. Conclusions: The preliminary results of this study suggest that the combination of gemcitabine and nab-paclitaxel with LSTA-1 and durvalumab is safe and potentially induces tumour infiltrating lymphocytes and RECIST response in locally advanced pancreatic adenocarcinomas. Clinical trial information: ACTRN12623000223639 .

Interaction and cleavage of cell and plasma proteins by the platelet-aggregating serine protease PA-BJ of Bothrops jararaca venom.

PA-BJ is a serine protease present in Bothrops jararaca venom that triggers platelet aggregation and granule secretion by activating the protease-activated receptors PAR-1 and PAR-4, without clotting fibrinogen. These receptors also have a relevant role in endothelial cells, however, the interaction of PA-BJ with other membrane-bound or soluble targets is not known. Here we explored the activity of PA-BJ on endothelial cell receptor, cytoskeleton, and coagulation proteins in vitro, and show the degradation of fibrinogen and protein C, and the limited proteolysis of actin, EPCR, PAR-1, and thrombomodulin. Antithrombin, factors XI and XIII and protein S were not cleaved by PA-BJ. Moreover, using surface plasmon resonance PA-BJ was demonstrated to bind to actin, EPCR, fibrinogen, PAR-1, and thrombomodulin, with dissociation constants (KD) in the micromolar range. Considering that these proteins play critical roles in pathways of blood coagulation and maintenance of endothelium integrity, their binding and cleavage by PA-BJ could contribute to the alterations in hemostasis and cell permeability observed in B. jararaca envenomation process.

Endothelial microvesicles in peripheral blood of pregnant women with preeclampsia

BACKGROUND: Endothelial dysfunction is the leading pathogenetic factor of preeclampsia. The function of the endothelium may be reflected in its ability to form microvesicles, which are generated by cells through the regulated shedding of the plasma membrane. AIM: The aim of this study was to evaluate the endothelial microvesicles count in peripheral blood of women with normal pregnancy and pregnancy complications such as gestational arterial hypertension and severe preeclampsia. MATERIALS AND METHODS: This study included 72 individuals, of whom there were healthy non-pregnant women (n = 21), women with normal pregnancy (n = 20), pregnant women with gestational arterial hypertension (n = 24), and pregnant women with severe preeclampsia (n = 7). To isolate microvesicles from peripheral blood, the differential centrifugation method was used. Microvesicles were treated with antibodies to vascular endothelial growth factor receptors (VEGFR1, VEGFR2), CD41a, CD34, and CD31 conjugated to fluorochromes. The absolute and relative count of microvesicles, as well as the fluorescence intensity, were analyzed using a BD FACSCanto II cytofluorimeter. RESULTS: In normal pregnancy, the count of microvesicles with the VEGFR1+, VEGFR2+, CD31+, and CD34+ phenotype was increased compared to non-pregnant women. In gestational arterial hypertension compared to normal pregnancy, no differences were found in the endothelial microvesicles count and endothelial marker expression. In severe preeclampsia, the total microvesicles count and endothelial cell derived microvesicles count in the peripheral blood plasma decreased in comparison with normal pregnancy and gestational arterial hypertension. While the expression of endothelial markers such as VEGFR1, VEGFR2, and CD34 in microvesicles membranes in severe preeclampsia increased compared to normal pregnancy and gestational arterial hypertension. CONCLUSIONS: An increase in the endothelial microvesicles count in normal pregnancy may be associated with an increase in the vascular bed area due to placenta formation. A decrease in the endothelial microvesicles count in severe preeclampsia is associated with damage to the endothelium and disruption of its function. Increased expression of endothelial cell receptors on microvesicles in severe preeclampsia may reflect compensatory reactions of the endothelium during the above damage.

Inhibition of lysophosphatidic acid receptor 2 attenuates neonatal chronic lung disease in mice by preserving vascular and alveolar development

AimBronchopulmonary dysplasia (BPD) is a common morbidity in extremely premature infants. Previous studies demonstrated the important role of lysophosphatidic acid (LPA) in inflammation in BPD. However, the role of LPA and its receptors in hyperoxia-induced vascular malformations in BPD remains to be elucidated. Methods and resultsElevated plasma LPA levels were observed in mice with BPD compared to controls (792 vs. 607 ng/mL, p < 0.05). Inhibition of LPA signaling protected against hyperoxia-induced lung injury in neonatal mice, demonstrated by a 2.8-fold increase in pulmonary vascular density and a 14% reduction in alveolar enlargement. In vitro studies showed that LPA suppressed tube formation in human umbilical vein endothelial cells (HUVECs) by approximately 50%. LPA receptor 2 (LPA2) was identified as a functional LPA receptor in primary endothelial cells from the lungs of hyperoxic mice and in HUVECs under hyperoxic conditions. The LPA2 antagonist H2L5186303 enhanced the tube formation ability of HUVECs exposed to LPA, both under normoxia (4-fold) and hyperoxia (5-fold). Moreover, H2L5186303 significantly protected against hyperoxia-induced vascular malformation (2-fold) and improved alveolarization in neonatal mice (12% decrease in mean linear intercept, MLI). Early growth response 1 (EGR1) was characterized as a downstream target of LPA2, silencing EGR1 restored tube formation in HUVECs exposed to LPA and hyperoxia. ConclusionsOur in vitro and in vivo findings demonstrate that the inhibition of LPA/LPA2 signaling mitigates hyperoxia-induced pulmonary vascular malformations, suggesting the LPA/LPA2-dependent signaling pathway has therapeutic potential for extremely premature infants with BPD.

Mineralocorticoid Receptor in Endothelial Cells Contributes to Vascular Endothelial Growth Factor Receptor Inhibitor-Induced Vascular and Kidney Damage.

Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer patient survival by inhibiting tumor angiogenesis. However, VEGFRis induce treatment-limiting hypertension which has been associated with impaired vascular endothelial cell (EC) function and kidney damage. The mineralocorticoid receptor (MR) regulates blood pressure (BP) via its effects on the vasculature and the kidney. Thus, we interrogated the role of the MR in EC dysfunction, renal impairment, and hypertension in a mouse model of VEGFRi-induced hypertension using sorafenib. EC dysfunction in mesenteric arterioles was assessed by immunoblotting for phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. Renal damage was measured by assessing glomerular endotheliosis histologically. BP was measured using implanted radiotelemetry. Six days of sorafenib treatment significantly impaired mesenteric resistance vessel EC function, induced renal damage, and increased BP. Pharmacologic MR blockade with spironolactone prevented the sorafenib-induced decline in eNOS phosphorylation and renal glomerular endotheliosis, without affecting systolic BP (SBP) or diastolic BP. Mice with the MR knocked out specifically in ECs (EC-MR-KO) were protected from sorafenib-induced EC dysfunction and glomerular endotheliosis, whereas smooth muscle cell-specific MR (SMC-MR) knockout mice were not. Neither EC-MR nor SMC-MR knockout affected the degree to which sorafenib increased SBP or diastolic BP. These results reveal that the MR, specifically in EC but not in SMCs, is necessary for VEGFRi-induced renal and vascular injury. While ineffective at lowering SBP, these data suggest potential therapeutic benefits of MR antagonists, like spironolactone, to protect the vasculature and the kidneys from VEGFRi-induced injury.

17β-estradiol inhibits Notch1 activation in murine macrophage cell line RAW 264.7.

Macrophages are major effectors in regulating immune response and inflammation. The pro-inflammatory phenotype (M1) is induced by the activation of the Toll-like receptor 4 (TLR4) on the macrophage surface, which recognizes lipopolysaccharide (LPS), a component of Gram-negative bacterial wall, and by the binding of interferon-gamma (IFNγ), a cytokine released by activated T lymphocytes, to its receptor (IFNGR). Among the pathways activated by LPS/IFNγ is the Notch pathway, which promotes the M1 phenotype. Conversely, 17β-estradiol (E2) has been shown to blunt LPS-mediated inflammatory response. While it has been shown that E2 regulates the activity of the Notch1 receptor in human endothelial cells, there is no evidence of estrogen-mediated regulation of Notch1 in macrophages. In this study, RAW 264.7 cells were stimulated with LPS/IFNγ in the presence or absence of E2 and/or N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of γ-secretase, the enzyme involved in Notch activation. The effects of treatment on inducible nitric oxide synthase (iNOS), on components of the Notch pathway, and MAPK (mitogen-activated protein kinase) were assessed by quantitative PCR and Western blotting. We found that E2, through a mechanism involving the inhibition of p38 phosphorylation, reduces the activation of Notch1 induced by LPS/IFNγ. On the contrary, Notch1 exerts a negative control on the estrogen receptor α (ERα) since Notch1 inhibition increases the protein levels of this receptor. In conclusion, we report for the first time a Notch-ERα interaction in macrophages. Our data suggest that E2 may reduce LPS/IFNγ-mediated M1 pro-inflammatory phenotype in macrophages by inhibiting Notch1. This finding encourages further studies on Notch1 inhibitors as novel treatments for inflammation-related diseases.

Small molecule modulation of insulin receptor-insulin like growth factor-1 receptor heterodimers in human endothelial cells

ObjectivesThe insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of two extracellular α-subunits and two transmembrane β -subunits. Insulin αβ and insulin like growth factor-1 αβ hemi-receptors can heterodimerize to form hybrids composed of one IR αβ and one IGF-1R αβ. The function of hybrids in the endothelium is unclear. We sought insight by developing a small molecule capable of reducing hybrid formation in endothelial cells. MethodsWe performed a high-throughput small molecule screening, based on a homology model of the apo hybrid structure. Endothelial cells were studied using western blotting and qPCR to determine the effects of small molecules that reduced hybrid formation. ResultsOur studies unveil a first-in-class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effects on IR or IGF-1R. This small molecule reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, increased basal phosphorylation of the downstream target Akt and enhanced insulin/insulin-like growth factor-1 and shear stress-induced serine phosphorylation of Akt. In primary saphenous vein endothelial cells (SVEC) from patients with type 2 diabetes mellitus undergoing coronary artery bypass (CABG) surgery, hybrid receptor expression was greater than in patients without type 2 diabetes mellitus. The small molecule significantly reduced hybrid expression in SVEC from patients with type 2 diabetes mellitus. ConclusionsWe identified a small molecule that decreases the formation of IR: IGF-1R hybrid receptors in human endothelial cells, without significant impact on the overall expression of IR or IGF-1R. In HUVECs, reduction of IR: IGF-1R hybrid receptors leads to an increase in insulin-induced serine phosphorylation of the critical downstream signalling kinase, Akt. The underpinning mechanism appears, at least in part to involve the attenuation of the inhibitory effect of IR: IGF-1R hybrid receptors on PI3-kinase signalling.

Apelin modulates inflammation and leukocyte recruitment in experimental autoimmune encephalomyelitis

Demyelination due to autoreactive T cells and inflammation in the central nervous system are principal features of multiple sclerosis (MS), a chronic and highly disabling human disease affecting brain and spinal cord. Here, we show that treatment with apelin, a secreted peptide ligand for the G protein-coupled receptor APJ/Aplnr, is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Apelin reduces immune cell entry into the brain, delays the onset and reduces the severity of EAE. Apelin affects the trafficking of leukocytes through the lung by modulating the expression of cell adhesion molecules that mediate leukocyte recruitment. In addition, apelin induces the internalization and desensitization of its receptor in endothelial cells (ECs). Accordingly, protection against EAE major outcomes of apelin treatment are phenocopied by loss of APJ/Aplnr function, achieved by EC-specific gene inactivation in mice or knockdown experiments in cultured primary endothelial cells. Our findings highlight the importance of the lung-brain axis in neuroinflammation and indicate that apelin targets the transendothelial migration of immune cells into the lung during acute inflammation.

Hyaluronic acid modified indocyanine green nanoparticles: a novel targeted strategy for NIR-II fluorescence lymphatic imaging.

The lymphatic system, alongside blood circulation, is crucial for maintaining bodily equilibrium and immune surveillance. Despite its importance, lymphatic imaging techniques lag behind those for blood circulation. Fluorescence imaging, particularly in the near-infrared-II (NIR-II) region, offers promising capabilities with centimeter-scale tissue penetration and micron-scale spatial resolution, sparking interest in visualizing the lymphatic system. Although indocyanine green (ICG) has been approved by the Food and Drug Administration (FDA) for use as a near-infrared-I (NIR-I) region fluorescent dye, its limitations include shallow penetration depth and low signal-to-noise ratio. Research suggests that ICG's fluorescence emission tail in the second near-infrared window holds potential for high-quality NIR-II imaging. However, challenges like short circulation half-life and concentration-dependent aggregation hinder its wider application. Here we developed HA@ICG nanoparticles (NPs), a superior ICG-based NIR-II fluorescent probe with excellent biocompatibility, prolonging in vivo imaging, and enhancing photostability compared to ICG alone. Leveraging LYVE-1, a prominent lymphatic endothelial cell receptor that binds specifically to hyaluronic acid (HA), our nanoprobes exhibit exceptional performance in targeting lymphatic system imaging. Moreover, our findings demonstrate the capability of HA@ICG NPs for capillary imaging, offering a means to assess local microcirculatory blood supply. These compelling results underscore the promising potential of HA@ICG NPs for achieving high-resolution bioimaging of nanomedicines in the NIR-II window.

Bacterial Opsonization Changes Adhesion Interactions between Endothelial Cells and Neutrophils in a Model of Experimental Septicemia.

The influence of non-opsonized and opsonized S. aureus 2879M and E. coli 321 strains on the total strength of interaction between the endothelial cell and neutrophil during the docking process was studied using in vitro model of experimental septicemia. We observed a decrease in the force and work of adhesion between receptors of neutrophils and endothelial cells under the influence of non-opsonized strains and further decrease in the affinity of single interactions between cells under the influence of opsonized S. aureus, which was compensated by an increase in the number of contacts, as well as an increase in the force of adhesion under the influence of opsonized E. coli compared to non-opsonized bacteria, which remained below the control level, while adhesion work reaches the control level. Thus, opsonization of S. aureus aggravates the "immunological uncoupling" between neutrophils and endothelial cells, while opsonization of E. coli reduces the pathological effect compared to non-opsonized bacteria.

Deletion of the Aryl Hydrocarbon Receptor in Endothelial Cells Improves Ischemic Angiogenesis in Chronic Kidney Disease

Background: Chronic kidney disease (CKD) is a strong yet underappreciated risk factor for peripheral arterial disease (PAD) that exacerbates disease progression and increases risk for limb amputation and cardiovascular mortality. An estimated 25-35% of CKD patients develop PAD, but mechanistic data linking these two pathologies is limited. Several tryptophan-derived uremic metabolites are retained in the blood and tissues as a consequence of CKD and are endogenous ligands of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that has been shown to regulate angiogenesis. Hypothesis: We tested the hypothesis that endothelial cell-specific conditional deletion of the AHR would enhance ischemic limb outcomes in mice with CKD and femoral artery ligation (FAL). Methods: Twelve-week-old male and female endothelium-specific AHR knockout mice (AHRecKO) and AHR floxed mice (AHRfl/fl, Cre-negative control) were fed 0.2% adenine to induce CKD or casein control diet (n=10/group/sex) prior to undergoing FAL to induce limb ischemia. Laser Doppler flowmetry was used to measure limb perfusion. Isometric contraction of the tibialis anterior in-situ via nerve stimulus was performed to measure muscle function. Skeletal muscle mitochondrial respiratory function and H2O2 production were evaluated. Primary endothelial cells were isolated from male and female wildtype mice and treated with known AHR ligand indoxyl sulfate (IS) under hypoxic conditions to uncover sex-dependent differences in AHR activating potential. Results: In male mice with CKD, endothelium-specific ablation of the AHR resulted in significant increases in percentage of perfused capillaries within the ischemic limb (AHRecKO 73.41% ± 4.80% vs AHRfl/fl 64.75% ± 8.325%; p= 0.0074), improved laser Doppler perfusion recovery in the paw and gastrocnemius muscle, and larger myofiber cross-sectional area ( p=0.0258) compared to AHRfl/fl mice. However, these improvements did not translate to improved muscle contractile function. In contrast, deletion of the AHR in female mice with CKD had no significant impact on major outcome measures. Primary cells from male mice treated with IS had significantly higher expression of the AHR controlled gene Cyp1a1 ( p=0.026) compared to cells from female mice. Conclusion: Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. These data reveal sex-dependent differences in AHR activating potential within endothelial cells that exist in the absence of sex hormones. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 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.

Abstract 1071: TGFβ Signaling Negatively Regulates Cardiac Lymphangiogenesis Following Myocardial Ischemia

Cardiovascular disease is the leading cause of death worldwide. Despite high prevalence, standard treatments have made little progress, and developing novel therapeutic treatments to improve cardiac diseases is the top priority of the field. Lymphatics are essential in maintaining tissue-fluid homeostasis, clearing cell debris, and trafficking immune cells. Over recent years, cardiac lymphatics have emerged as a potential target for treatment of cardiovascular diseases, as therapeutic lymphangiogenesis is shown to improve cardiac function following myocardial infarction (MI). However, pathologic lymphangiogenesis induced by MI is reported to be dysfunctional, suggesting cellular or molecular changes occurring in MI directly impact the structure and function of cardiac lymphatics. However, the underlying mechanisms leading to these changes have not been fully explored. In this study we propose TGFβ, a profibrotic signaling molecule increased in ischemic heart diseases, negatively regulates cardiac lymphangiogenesis and lymphatic function following MI. We generated mice with conditional deletion of TGFβ receptor in Lymphatic endothelial cells (LECs) and found that these conditional null (LEC-DKO) mice showed improved post-MI cardiac function and increased lymphangiogenesis compared to controls. Mechanistically, we found that LECs with TGFβ treatment showed increased Endothelial to Mesenchymal Transition (EndMT), and impaired junction integrity. Bulk RNAseq results confirmed these findings as genes involved in EndMT were highly upregulated, and LEC marker related genes were significantly downregulated in LECs in the presence of TGFβ. More importantly, GSEA revealed metabolic reprogramming in LECs treated with TGFβ, with significantly increased expression in glycolysis related genes, but significant downregulation of genes related to fatty acid oxidation. These data provide a possible mechanism by which TGFβ signaling induces LEC metabolic switch to impede lymphangiogenesis and promote cardiac dysfunction following MI, and inhibition of TGFβ signaling in LECs could provide a novel therapeutic strategy to improve lymphatic vascular integrity and lymphangiogenesis, thereby improving cardiac function following MI.

A Review: The Significance of Toll-Like Receptors 2 and 4, and NF-κB Signaling in Endothelial Cells during Atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory vascular disease that begins with endothelial activation followed by a series of inflammatory responses, plaque formation, and finally rupture. An early event in endothelial dysfunction is activation of the nuclear factor-κB (NF-κB) signaling axis. Toll-like receptors (TLRs) in endothelial cells (ECs) play an essential role in recognizing pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and lifestyle-associated molecular patterns (LAMPs). Activation of the canonical NF-κB pathway stimulates the expression of cytokines, chemokines, and an array of additional genes which activate and amplify AS-associated inflammatory responses. In this review, we discuss the involvement of TLR2/4 and NF-κB signaling in ECs during AS initiation, as well as regulation of the inflammatory response during AS by noncoding RNAs, especially microRNA (miRNA) and circular RNA (circRNA).

Blood-brain barrier disruption and edema formation due to prolonged starvation in wild-type mice.

Different types of diseases have been treated by restricted caloric intake or fasting. Although during this long time, fasting protective measures, for example, supplements, are given to the patients to protect vital organs such as the liver and kidney, little attention is given to the brain. The current research aims to investigate hypoglycemia due to prolonged fasting disrupts blood-brain barrier (BBB) in mice. Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques were used to examine the expression of different genes. Evans blue extravasation and wet-dry technique were performed to evaluate the integrity of BBB and the formation of brain edema, respectively. We confirmed that hypoglycemia affected mice fasting brain by examining the increased expression of glucose transporter protein 1 and hyperphosphorylation of tau protein. We subsequently found downregulated expression of some genes, which are involved in maintaining BBB such as vascular endothelial growth factor (VEGF) in astrocytes and claudin-5 (a vital component of BBB) and VEGF receptor (VEGFR1) in endothelial cells by ISH. We also found that prolonged fasting caused the brain endothelial cells to express lipocalin-2, an inflammatory marker of brain endothelial cells. We performed Evans blue extravasation to show more dye was retained in the brain of fasted mice than in control mice as a result of BBB disruption. Finally, wet-dry method showed that the brain of prolonged fasted mice contained significantly higher amount of water confirming the formation of brain edema. Therefore, special attention should be given to the brain during treatment with prolonged fasting for various diseases. Our results demonstrated that hypoglycemia due to prolonged fasting disrupts BBB and produces brain edema in wild-type mice, highlighting the importance of brain health during treatment with prolonged fasting.

Proteoglycans of basement membranes: Crucial controllers of angiogenesis, neurogenesis, and autophagy.

Anti-angiogenic therapy is an established method for the treatment of several cancers and vascular-related diseases. Most of the agents employed target the vascular endothelial growth factor A, the major cytokine stimulating angiogenesis. However, the efficacy of these treatments is limited by the onset of drug resistance. Therefore, it is of fundamental importance to better understand the mechanisms that regulate angiogenesis and the microenvironmental cues that play significant role and influence patient treatment and outcome. In this context, here we review the importance of the three basement membrane heparan sulfate proteoglycans (HSPGs), namely perlecan, agrin and collagen XVIII. These HSPGs are abundantly expressed in the vasculature and, due to their complex molecular architecture, they interact with multiple endothelial cell receptors, deeply affecting their function. Under normal conditions, these proteoglycans exert pro-angiogenic functions. However, in pathological conditions such as cancer and inflammation, extracellular matrix remodeling leads to the degradation of these large precursor molecules and the liberation of bioactive processed fragments displaying potent angiostatic activity. These unexpected functions have been demonstrated for the C-terminal fragments of perlecan and collagen XVIII, endorepellin and endostatin. These bioactive fragments can also induce autophagy in vascular endothelial cells which contributes to angiostasis. Overall, basement membrane proteoglycans deeply affect angiogenesis counterbalancing pro-angiogenic signals during tumor progression, and represent possible means to develop new prognostic biomarkers and novel therapeutic approaches for the treatment of solid tumors.

Effects of Ghrelin on Plasminogen Activator Activity in Human Umbilical Vein Endothelial Cells

Ghrelin and its growth hormone secretagogue receptor (GHSR) have been found in the placenta, both in endothelial and trophoblast cells. Ghrelin has been shown to decrease blood pressure in several systems and improve endothelial function by stimulating VEGF production. Because locally increased Ghrelin was detected in the preeclamptic fetoplacental unit, we hypothesized its involvement in the fibrinolysis and vascular tone typically observed in preeclamptic patients. This study aimed to evaluate the synthesis of plasminogen activators (PAs), PA inhibitor-1 (PAI-1), and urokinase-type PA (uPA) receptor (uPAR) in human umbilical vein endothelial cells (HUVECs) since the components of the PA/plasmin system are vital players in the extracellular matrix remodeling process necessary for angiogenesis. HUVECs were treated for 24 h with increasing concentrations of Ghrelin (10−11–10−7 M) or IL-1β (0.1 ng/mL). PAs, PAI-1, and uPAR mRNAs were determined by real-time PCR and PA activity was determined by casein underlay. We demonstrated an increase in uPA, tissue-type PA (tPA), and uPAR mRNA; a reduction in PAI-1 mRNA in HUVECs treated with Ghrelin; and an increase in total uPA activity. In conclusion, our results suggest a potential compensatory physiological mechanism for Ghrelin in response to the maternal endothelial dysfunction observed in the preeclamptic fetoplacental unit.

Identification of Neural (N)-Cadherin as a Novel Endothelial Cell Receptor for Fibrin and Localization of the Complementary Binding Sites.

Based on the high structural homology between vascular endothelial (VE)-cadherin and neural (N)-cadherin, we hypothesized that fibrin, which is known to interact with VE-cadherin and promote angiogenesis through this interaction, may also interact with N-cadherin. To test this hypothesis, we prepared fibrin and its plasmin-produced and recombinant fragments covering practically all parts of the fibrin molecule. We also prepared the soluble extracellular portion of N-cadherin (sN-cadherin), which includes all five extracellular N-cadherin domains, and studied its interaction with fibrinogen, fibrin, and the aforementioned fibrin fragments using two independent methods, ELISA and SPR. The experiments confirmed our hypothesis, revealing that fibrin interacts with sN-cadherin with high affinity. Furthermore, the experiments localized the N-cadherin binding site within the fibrin βN-domains. Notably, the recombinant dimeric (β15-66)2 fragment, corresponding to these domains and mimicking their dimeric arrangement in fibrin, preserved the N-cadherin-binding properties of fibrin. To localize the fibrin binding site within N-cadherin, we performed ELISA and SPR experiments with (β15-66)2 and recombinant N-cadherin fragments representing its individual extracellular domains and combinations thereof. The results obtained indicate that the interaction of fibrin with N-cadherin occurs through the third and fifth extracellular domains of the latter. This is in contrast to our previous study, which revealed that fibrin interacts only with the third extracellular domain of VE-cadherin. In conclusion, our study identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within both fibrin and N-cadherin. The pathophysiological role of this interaction remains to be established.

Arsenic Trioxide Decreases Lymphangiogenesis by Inducing Apoptotic Pathways and Inhibition of Important Endothelial Cell Receptors.

Tumor-induced lymphangiogenesis is strongly associated with the formation of tumor metastasis. Therefore, the regulation of lymphangiogenesis offers a promising target in cancer therapy. Arsenic trioxide (ATO) is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). As ATO mediates anti-angiogenic effects on endothelial and tumor cells, we aimed to explore the impact of ATO on lymphangiogenesis in human lymphatic endothelial cells (LEC). The BrdU assay and flow cytometry analysis were used to evaluate the influence of ATO on the proliferation and cell cycle distribution of LECs. The lymphatic suppression effects of ATO were investigated in vitro using the lymphatic tube formation assay. The effects of ATO on apoptosis, mitochondrial membrane potential and endothelial cell receptors were investigated by Western blotting, ELISA, flow cytometry and qRT-PCR. The treatment of LECs with ATO attenuated cell proliferation, blocked tube formation and induced subG0/G1 arrest in LECs, thus suggesting enhanced apoptosis. Although subG0/G1 arrest was accompanied by the upregulation of p21 and p53, ATO treatment did not lead to visible cell cycle arrest in LECs. In addition, ATO caused apoptosis via the release of cytochrome c from mitochondria, activating caspases 3, 8 and 9; downregulating the anti-apoptotic proteins survivin, XIAP and cIAP-2; and upregulating the pro-apoptotic protein Fas. Furthermore, we observed that ATO inhibited the VEGF-induced proliferation of LECs, indicating that pro-survival VEGF/VEGFR signaling was affected by ATO treatment. Finally, we found that ATO inhibited the expression of the important endothelial cell receptors VEGFR-2, VEGFR-3, Tie-2 and Lyve-1. In conclusion, we demonstrate that ATO inhibits lymphangiogenesis by activating apoptotic pathways and inhibiting important endothelial cell receptors, which suggests that this drug should be further evaluated in the treatment of tumor-associated lymphangiogenesis.

Anti‐mouse CD98hc nanobodies for brain delivery of a therapeutic moiety

AbstractBackgroundThe blood‐brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders such as Alzheimer’s disease. Interestingly, monoclonal antibodies generated against receptor‐mediated transcytosis receptors are able to transport therapeutic proteins across the BBB. Nanobodies, which are the variable domain isolated from camelid heavy‐chain only antibodies have also shown their potential to deliver therapeutics into the CNS. Here we aimed to expand the panel of nanobodies targeting other brain endothelial cell receptors.MethodCamelids were immunized with a pool of DNA encoding for several brain endothelial cell receptors. One target was chosen to validate the immunization and discovery approach, CD98hc, previously suggested to allow drug shuttling across the BBB. Nanobody libraries were selected on Chinese Hamster Ovarian (CHO) cells overexpressing the target of interest. Selected nanobodies were screened in vivo based on their potential to deliver into the CNS either a biologically active neuropeptide neurotensin to reduce body temperature, or a BACE1 inhibiting antibody that reduces the accumulation of Aβ1‐40 species.ResultDNA immunization followed by selections on CHO cells overexpressing the target of interest resulted in specific anti‐mouse CD98hc binders with affinities in the low nanomolar range. In vivo screening of mouse CD98hc nanobody binders revealed some discrepancy between the two methods used. Whereas none of the tested nanobodies fused to neurotensin was able to reduce body temperature, nanobodies fused to the anti‐BACE1 antibody 1A11 effectively reduced Aβ1‐40 levels in the brain. We suggest that the neurotensin approach, although having shown robustness with anti‐TfR nanobodies, might lack sensitivity when screening non‐TfR binders. We suggest BACE1 inhibition as a more robust model to validate CD98hc‐mediated transport across the BBB.ConclusionThis is a proof of concept study in which we have optimized our nanobody discovery approach to find nanobodies against BBB targets, and validated CD98hc as a receptor to target in order to shuttle drugs into the brain. In the future we will explore whether nanobodies raised against novel brain endothelial cell receptors could also improve brain penetration of biologics.

Ultrasound imaging of renal fibrosis using gold nanoparticles

Abstract Introduction Microbubble ultrasound contrast agents have limitations due to their short circulation half-life and poor acoustic attenuation. In our previous study, pH-responsive gold nanoparticles (2∼3 nm) were optimized for ultrasound fluorescence molecular imaging of mouse kidneys. Renal fibrosis is associated with increased expression of the integrin αvβ3 receptor in neovascular endothelial cells. To address this, gold nanoparticles (cRGD-GSH-AuNPs) were synthesized by introducing RGD peptide, which specifically binds to integrin. Purpose The purpose of this study was to evaluate the potential of cRGD-GSH-AuNPs as a contrast agent for targeted ultrasound imaging of renal fibrosis. Methods Cyclic arginine-glycine-aspartic (cRGD) targeting integrin αvβ3 receptors was linked with glutathione (GSH)-modified gold nanoparticles (AuNPs) to create cRGD-GSH-AuNPs. After characterizing their physical properties and verifying their biocompatibility, GSH-AuNPs and cRGD-GSH-AuNPs were injected into mice with the renal fibrosis model. The renal imaging effect was evaluated by ultrasound and fluorescence imaging, and the targeting of the particles was confirmed by pathological tissue staining. Results The average size of cRGD-GSH-AuNPs determined by electron microscopy was 1.7 ± 0.2 nm, and the particles maintained biological stability after modification. The average ultrasound intensity of cRGD-GSH-AuNPs in the renal fibrosis group (22 ± 2 dB) was significantly higher than that of GSH-AuNPs in the fibrosis group (3.5 ± 3 dB), cRGD-GSH-AuNPs in the control group (1.5 ± 1 dB), and GSH-AuNPs in the control group (2 ± 1.5 dB) (all, P &amp;lt; 0.05). Intravenous injection of cRGD-GSH-AuNPs significantly enhanced the ultrasound signal intensity in fibrotic kidneys (see Figure 1). Conclusions Gold nanoparticles targeting integrin αVβ3 receptors achieve specific non-invasive imaging of renal fibrosis, and are a promising ultrasound contrast agent for the evaluation of renal fibrosis.Figure 1