Soluble Growth Research Articles

Virtual Screening of Protein Data Bank via Docking Simulation Identified the Role of Integrins in Growth Factor Signaling, the Allosteric Activation of Integrins, and P-Selectin as a New Integrin Ligand.

Integrins were originally identified as receptors for extracellular matrix (ECM) and cell-surface molecules (e.g., VCAM-1 and ICAM-1). Later, we discovered that many soluble growth factors/cytokines bind to integrins and play a critical role in growth factor/cytokine signaling (growth factor-integrin crosstalk). We performed a virtual screening of protein data bank (PDB) using docking simulations with the integrin headpiece as a target. We showed that several growth factors (e.g., FGF1 and IGF1) induce a integrin-growth factor-cognate receptor ternary complex on the surface. Growth factor/cytokine mutants defective in integrin binding were defective in signaling functions and act as antagonists of growth factor signaling. Unexpectedly, several growth factor/cytokines activated integrins by binding to the allosteric site (site 2) in the integrin headpiece, which is distinct from the classical ligand (RGD)-binding site (site 1). Since 25-hydroxycholesterol, a major inflammatory mediator, binds to site 2, activates integrins, and induces inflammatory signaling (e.g., IL-6 and TNFα secretion), it has been proposed that site 2 is involved in inflammatory signaling. We showed that several inflammatory factors (CX3CL1, CXCL12, CCL5, sPLA2-IIA, and P-selectin) bind to site 2 and activate integrins. We propose that site 2 is involved in the pro-inflammatory action of these proteins and a potential therapeutic target. It has been well-established that platelet integrin αIIbβ3 is activated by signals from the inside of platelets induced by platelet agonists (inside-out signaling). In addition to the canonical inside-out signaling, we showed that αIIbβ3 can be allosterically activated by inflammatory cytokines/chemokines that are stored in platelet granules (e.g., CCL5, CXCL12) in the absence of inside-out signaling (e.g., soluble integrins in cell-free conditions). Thus, the allosteric activation may be involved in αIIbβ3 activation, platelet aggregation, and thrombosis. Inhibitory chemokine PF4 (CXCL4) binds to site 2 but did not activate integrins, Unexpectedly, we found that PF4/anti-PF4 complex was able to activate integrins, indicating that the anti-PF4 antibody changed the phenotype of PF4 from inhibitory to inflammatory. Since autoantibodies to PF4 are detected in vaccine-induced thrombocytopenic thrombosis (VIPP) and autoimmune diseases (e.g., SLE, and rheumatoid arthritis), we propose that this phenomenon is related to the pathogenesis of these diseases. P-selectin is known to bind exclusively to glycans (e.g., sLex) and involved in cell-cell interaction by binding to PSGL-1 (CD62P glycoprotein ligand-1). Unexpectedly, through docking simulation, we discovered that the P-selectin C-type lectin domain functions as an integrin ligand. It is interesting that no one has studied whether P-selectin binds to integrins in the last few decades. The integrin-binding site and glycan-binding site were close but distinct. Also, P-selectin lectin domain bound to site 2 and allosterically activated integrins.

The influence of IGF-I and Emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model

Aim: Tooth decay and associated periodontal disease remain the most common chronic diseases in current society. In the future, individually tailored, more effective therapeutic treatment options could be provided through the application of tissue engineering and regenerative medicine and dentistry (TERMD) approaches. A relevant finding is the potential of both dental mesenchymal and dental epithelial cells to regenerate mineralized dentin and enamel tissues, respectively.Materials and Methods: In the current study, a multi-layered, bioengineered tooth bud model was assembled by combining human dental mesenchymal (hDM) and porcine dental epithelial cells (pDE). The hDM cells were seeded onto poly(epsilon-caprolactone)/ poly (lactide-co-glycolide) (PCL/PLGA) wet electro-spun scaffolds overlayed with confluent pDE cell sheet harvested from thermo-reversible tissue culture plates. The so-formed multi-layered bioengineered tooth bud was then used to study the mineralization potential of the dental cells in in vitro culture. It was hypothesized that the addition of the in media soluble factors Insulin-like growth factor 1 (IGF-I) and the extracellular matrix derivate Emdogain (EMD) would result in enhanced differentiation and mineralized dental tissue formation. Scanning Electron morphological observation was used to characterize scaffolds porosity. Histological and immunofluorescent analyses confirmed the localization of hDM cells inside the scaffold, an intact pDE cell sheet, and the presence of beta-integrin 1-positive cell-cell junctions connecting the two.Results: Scanning Electron Microscopy showed that EMD, in particular, enhanced the mineralization potential of pDE cells. qRT-PCR analyses showed that both EMD and IGF-1 significantly enhanced the expression of Ameloblastin (AMBN), reflecting pDE cell differentiation.Conclusion: In conclusion, these results proved the hypothesis that both EMD and IGF-1 should be considered for their utility in preclinical dental tissue engineering approaches.

The feasibility of soluble Fms-Like Tyrosine kinase-1 (sFLT-1) and Placental Growth Factor (PlGF) ratio biomarker in predicting preeclampsia and adverse pregnancy outcomes among medium to high risk mothers in Kuala Lumpur, Malaysia.

Preeclampsia significantly contributes to maternal and perinatal morbidity and mortality. It is imperative to identify women at risk of developing preeclampsia in the effort to prevent adverse pregnancy outcomes through early intervention. Soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) level changes are noticeable several weeks before the onset of preeclampsia and its related complications. This study evaluated the feasibility of the sFlt-1/PlGF biomarker ratio in predicting preeclampsia and adverse pregnancy outcomes using a single cut-off point of >38. This is a prospective cohort study conducted at a single tertiary centre, in an urban setting in Kuala Lumpur, Malaysia, between December 2019 and April 2021. A total of 140 medium to high risk mothers with singleton pregnancies were recruited at ≥20 weeks' gestation. sFlt-1/PlGF ratio was measured and the participant monitored according to a research algorithm until delivery. The primary outcome measure was incidence of preeclampsia and the secondary outcome measure was incidence of other adverse pregnancy outcomes. The overall incidence of preeclampsia was 20.7% (29/140). The mean sFlt-1/PlGF ratio was significantly higher in preeclampsia (73.58 ± 93.49) compared to no preeclampsia (13.41 ± 21.63) (p = 0.002). The risk of preeclampsia (adjusted OR 28.996; 95% CI 7.920-106.164; p<0.001) and low Apgar score (adjusted OR 17.387; 95% CI 3.069-98.517; p = 0.028) were significantly higher among women with sFlt-1/PlGF ratio >38 compared with sFLT-1/PlGF ratio ≤38. The area under the receiver-operator characteristic curve (AUC) for a combined approach (maternal clinical characteristics and biomarker) was 86.9% (p<0.001, 95% CI 78.7-95.0) compared with AUC biomarker alone, which was 74.8% (p<0.001, 95% CI 63.3-86.3) in predicting preeclampsia. The test sensitivity(SEN) was 58.6%, specificity (SPEC) 91%,positive predictive value (PPV) 63% and negative predictive value (NPV) 89.3% for prediction of preeclampsia. For predicting a low Apgar score at 5 minutes, the SEN was 84.6%, SPEC 87.4%, PPV 40.7%, and NPV 98.2%; low birth weight with SEN 52.6%,SPEC 86.0%, PPV 37.0%, NPV 92.0%; premature delivery with SEN 48.5%, SPEC 89.5%, PPV 59.3%, NPV 84.7% and NICU admission with SEN 50.0%, SPEC 85.8%, PPV 37.0% and NPV 91.2%. It is feasible to use single cut-off point of >38 ratio of the biomarkers sFlt-1/PlGF in combination with other parameters (maternal clinical characteristics) in predicting preeclampsia and adverse pregnancy outcomes among medium to high risk mothers without restricting outcome measurement period to 1 and 4 weeks in a single urban tertiary centre in Kuala Lumpur, Malaysia.

Isoforms of soluble vascular endothelial growth factor in stress-related mental disorders: a cross-sectional study

Vascular endothelial growth factor (VEGF) has been implicated in the pathophysiology of stress-related mental disorders. However, VEGF levels have seldom been compared across mental disorders and never by isoforms. Pathophysiological processes involving leakage of astrocyte-derived extracellular vesicles (EVs) across the blood–brain barrier could be associated with VEGF levels in patients with stress-related mental disorders. This cross-sectional study compared plasma levels of VEGF121, VEGF165, and VEGF121 + VEGF165 (VEGFtotal) in patients with stress-induced exhaustion disorder (SED) (n = 31), patients with major depressive disorder (MDD) (n = 31), and healthy controls (n = 61). It also analyzed the correlation between VEGF and astrocyte-derived EVs in plasma. An enzyme-linked immunosorbent assay (ELISA) was used to measure VEGF121 and VEGF165 in citrate plasma, and flow cytometry was used to measure astrocyte-derived EVs in plasma. The mean concentration of soluble VEGF121 (sVEGF121) was significantly higher in patients with SED than healthy controls (P = 0.043). Mean sVEGF165 was significantly lower in patients with MDD than patients with SED (P = 0.004) or healthy controls (P = 0.037). Mean sVEGFtotal was significantly higher in patients with SED than in patients with MDD (P = 0.021) and also higher in patients with SED than healthy controls (P = 0.040). Levels of sVEGF121 were positively correlated with levels of astrocyte-derived EVs only in patients with SED (P = 0.0128). The same was true of levels of sVEGFtotal and astrocyte-derived EVs (P = 0.0046). Differing levels of VEGF isoforms may reflect different pathophysiological mechanisms in SED and MDD. Further research is needed to better understand the potential roles of VEGF isoforms and astrocyte-derived EVs in mental disorders.

Abstract 1839: Nanobodies against EGF enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer cell models

Abstract Background: The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGFR mutations. However, acquired resistance appears invariably due to several mechanisms. The strategy of using EGF-targeted nanobodies (Nbs) to block the initial step of the EGFR pathway constitutes a new research area. Nbs also offer several advantages compared to traditional mAbs, such as their reduced size, higher stability and tissue penetration, low production costs and lack of Fc effector function, which provide key advantages for targeting soluble tumoral growth factors. In this study we investigated the efficacy of anti-EGF Nbs to reduce osimertinib-resistance. Experimental procedures: Cell viability and colony formation assays were performed in PC9 and PC9-osimertinib-resistant NSCLC cell lines tested with anti-EGF Nbs alone or in combination with osimertinib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study protein expression and activation. Summary of data: Two anti-EGF Nbs, generated in our laboratory, inhibited cell viability and colony formation in PC9 and PC9-derived osimertinib-resistant cell lines. The combination of these Nbs with osimertinib improved the antitumoral efficacy of this EGFR-TKI in cell viability and colony formation experiments. The combination of anti-EGF Nbs with osimertinib clearly decreases the activation of proteins involved in the EGFR pathway, such as, EGFR, Akt and Erk 1/2. In addition, it increases cellular apoptosis, and decreases the expression of Hes1, a marker of cancer stem cells involved in metastasis and osimertinib resistance mechanisms. Conclusions: We conclude that the combination of anti-EGF nanobodies and osimertinib exhibits a good efficacy in inhibiting cell viability and colony formation, while, inhibiting expression and activation of proteins involved in osimertinib resistance. Citation Format: Salvador Guardiola, Macarena Sánchez-Navarro, Ernest Giralt, Rafael Rosell, Jordi Codony-Servat. Nanobodies against EGF enhance the antitumoral effect of osimertinib and overcome resistance in non-small cell lung cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1839.

Effect of bazedoxifene on expressions of VEGF, VEGFR2, COX-2 and inflammatory factors in a rat endometriosis model

Purpose: To study the effect of bazedoxifene on the expressions of vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor 2 (sVEGFR2), cyclooxygenase synthase 2 (COX-2) and inflammatory factors in a rat endometriosis (EMS) model.&#x0D; Methods: Thirty rats (EMS) were divided into untreated control group, bazedoxifene group and celecoxib group (10 rats per group). Bazedoxifene and celecoxib were administered at doses of 3 and 25 mg/kg, respectively. Negative control rats served as control, and were given 0.9 % sodium chloride at a dose of 10 mL/kg. All treatments were given intragastrically for 21 days. Levels of VEGF, sVEGFR2 and COX-2 in uterine ectopic endometrium were assayed.&#x0D; Results: The levels of VEGF and sVEGFR2 in the serum and peritoneal fluid of the untreated control group were significantly higher than the corresponding control values, while VEGF and sVEGFR2 levels in the serum and peritoneal fluid of the bazedoxifene group were significantly lower than those in the untreated control group (p &lt; 0.05). Bazedoxifene group had lower VEGFR2 and COX-2 levels than untreated control group (p &lt; 0.05). Expression of COX-2 protein in the ectopic endometrium of celecoxib group was significantly lower than that in the untreated control group (p &lt; 0.05).&#x0D; Conclusion: Bazedoxifene alleviates angiogenesis and inflammatory factors in serum and peritoneal fluid of EMS rats, inhibits the expression of sVEGFR2 and COX-2 in ectopic endometrium, and also inhibits the growth of ectopic endometrium. This finding may help to discover additional new drugs.

Early Serum Heme Oxygenase-1, Soluble Vascular Endothelial Growth Factor Receptor-1, and B-Cell Lymphoma/Leukemia-2 Levels and Unfavorable Obstetric Outcomes

OBJECTIVE: To examine early pregnancy levels of serum heme oxygenase-1, soluble vascular endothelial growth factor receptor-1, B-cell lymphoma/leukemia-2 in relation to unfavorable pregnancy outcomes, including preeclampsia, fetal growth restriction, spontaneous preterm birth, gestational diabetes mellitus and fetal macrosomia. STUDY DESIGN: A total of randomly selected 140 pregnancies were included in this prospective study. Peripheral blood samples were obtained between 110/7 and 136/7 gestational weeks. All pregnancies were followed up until the outcomes were obtained and classified as preeclampsia, fetal growth restriction, spontaneous preterm birth, gestational diabetes mellitus, fetal macrosomia, and uncomplicated ones. RESULTS: Significantly high levels of early serum heme oxygenase-1 were found in the cases who subsequently developed preeclampsia, spontaneous preterm birth, and fetal macrosomia (p&lt;0.05), in concordance with high soluble vascular endothelial growth factor receptor-1 levels of the preeclampsia group, however, B-cell lymphoma/leukemia-2 s were similar in all groups. As soluble vascular endothelial growth factor receptor-1 predicted preeclampsia at a value of 11.905 ng/mL, the cutoff values for the heme oxygenase-1 to predict preeclampsia, spontaneous preterm birth, and fetal macrosomia were 0.372 ng/mL, 0.354 ng/mL and 0.494 ng/ml, respectively. CONCLUSION: Elevated first trimester heme oxygenase-1 levels are remarkable in the pregnancies associated with preeclampsia, spontaneous preterm birth, and fetal macrosomia in comparison with uncomplicated ones.

Effects of exercise training with weight loss on skeletal muscle expression of angiogenic factors in overweight and obese older men.

Low skeletal muscle capillarization is associated with impaired glucose tolerance (IGT); however, aerobic exercise training with weight loss (AEX + WL) increases skeletal muscle capillarization and improves glucose tolerance in adults with IGT. Given that the expression of angiogenic growth factors mediates skeletal muscle capillarization, we sought to determine whether angiogenic growth factor levels are associated with low capillarization in those with IGT versus normal glucose tolerance (NGT) or to the benefits of AEX + WL in both groups. Sixteen overweight or obese men 50-75 yr of age completed 6 mo of AEX + WL with oral glucose tolerance tests and vastus lateralis muscle biopsies for measurement of muscle vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase receptor-1 (sFlt-1), and basic fibroblast growth factor (bFGF). At baseline, all growth factor levels were numerically lower in IGT than NGT, but these did not reach statistical significance (P = 0.06-0.33). Following AEX + WL, aerobic capacity [maximal oxygen consumption (V̇o2max)] increased by 16%, whereas body weight and 120-min postprandial glucose levels decreased by 10% and 15%, respectively (P ≤ 0.001 for all). There was a main effect of AEX + WL to increase VEGF (0.095 ± 0.016 vs. 0.114 ± 0.018 ng/µg, P < 0.05), PlGF (0.004 ± 0.001 vs. 0.005 ± 0.001 ng/µg, P < 0.05), and sFlt-1 (0.216 ± 0.029 vs. 0.264 ± 0.036 ng/µg, P < 0.01), with overall increases driven by the IGT group. These results suggest that 6 mo of AEX + WL increases skeletal muscle angiogenic growth factor levels in obese older adults with IGT and NGT, which may contribute to our previous findings that AEX + WL increases capillarization to improve glucose tolerance in those with IGT.NEW & NOTEWORTHY Skeletal muscle capillarization is lower in adults with impaired glucose tolerance than normal controls. This may, in part, be attributable to differential expression of angiogenic growth factors in skeletal muscle. Using a 6-mo aerobic exercise intervention with ∼10% body weight loss (AEX + WL), we show that the expression of angiogenic growth factors tends to be lower in adults with impaired glucose tolerance compared with normal controls and that AEX + WL increased expression of angiogenic growth factors in all participants.

The Value of Biomarkers in the Diagnosis and Prognosis of Heart Failure in Older Age

The search for reliable algorithms for diagnosing heart failure with preserved left ventricular ejection fraction (LVEF) in elderly patients is an urgent problem due to the low specificity of clinical manifestations and the peculiarities of involutive processes occurring in the human body. As an alternative diagnostic approach, it is possible to determine in the blood laboratory biochemical markers — a promising method of diagnosis, prognosis and control of the effectiveness of treatment. The article examines the significance of myocardial stress markers (brain natriuretic peptide, N-terminal brain natriuretic peptide, median fragment of atrial natriuretic peptide); «mechanical» myocardial stress (soluble stimulating growth factor expressed by gene 2 — sST2), copeptin, galectin-3 in patients with heart failure and preserved LVEF, including older persons, as well as the possibility of their use in outpatient practice to predict the course of heart failure. The contribution of the multimarker model for a comprehensive assessment of prognosis is discussed, taking into account both the «hemodynamic» side of myocardial stress (pressure or volume overload, markers — natriuretic peptides), and «mechanical» (fibrosis / hypertrophy / heart remodeling, marker — sST2) myocardial changes.

Engineering soluble artificial epidermal growth factor receptor mimics capable of spontaneous in vitro dimerization.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non-membrane-bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self-associate upon mixing. Two fusion protein species (P3-EGFR and P4-EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5-fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand-induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class.

Role of proteins in MAPK pathways on Cleft Lip Patients of Protomalayid Race in East Nusa Tenggara Province, Indonesia

Cleft lip is a disruption which happens in the upper lip joining process due to multifactorial causes such as genetic, nutritional, environmental, and socio-economic. East Nusa Tenggara is a province in Indonesia with Protomalayid race population that has a high incidence of cleft lip of 5-6 per 1000 births. This study therefore aims to determine the expression and relationship of proteins in the Mitogen-activated Protein Kinase (MAPK) pathway of cleft lip in the Protomalayid race of East Nusa Tenggara Province. The method used was Hematoxylin-Eosin staining and Immunohistochemical technique to identify the expression of Transforming Growth Factor Alpha (TGF-α) protein, Extracellular signal-regulated kinases (ERK1), Activator Protein (AP-1), Transforming Growth Factor Beta-1 (TGF-β1), p38, Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1), Epidermal Growth Factor Receptor (EGFR), and Fibroblast Growth Factor 2 (FGF- 2) in cleft lip cells. The result shows that the expressions of TGF-α, EGFR, FGF-2, and ERK-1 decreased, but that of AP-1, TGF-β1, p38, and SVEGFR1 increased. Meanwhile, the results of the Pearson correlation analysis showed the TGFα-ERK1, TGFβ1-p38, AP1-p38, EGFR-ERK1, and FGF2-p38 had a positive relationship. All the factors in the study have correlations (except FGF2 - ERK1). The relationship between FGF2-ERK1 was not significant, while SVEGFR1- p38 protein has a negative relationship.

Angiogenesis-related factors associated with nivolumab efficacy in patients with advanced gastric cancer after refractory or intolerant to ramucirumab-based therapy.

234 Background: Nivolumab (Nivo) is a standard therapy after ramucirumab (RAM)-based second-line chemotherapy in patients (pts) with advanced gastric cancer (AGC). However, the relationship between angiogenesis-related factors and the efficacies of immune-checkpoint inhibitors after anti-angiogenic chemotherapy is unclear. Methods: We retrospectively evaluated pts with AGCs who received Nivo after RAM-based second-line chemotherapy at a single institution from Nov 2017 to Aug 2019. Clinical benefit of Nivo was defined as CR, PR, or &gt;4 months of SD and non-CR/non-PD. In addition, preserved serum samples at time points of pre-RAM, pre-Nivo, and post-Nivo (within 12 weeks from the start of Nivo) were analyzed by using the designated panel, containing vascular endothelial growth factor-A/D (VEGF-A/D), placental growth factor (PlGF), soluble vascular endothelial growth factor receptor-1/2/3 (sVEGFR-1/2/3), Interleukin-6/8 (IL-6/8), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), angiopoietin-2, hepatocyte growth factor (HGF), interferon gamma (INF-γ), osteopontin (OPN), soluble neuropilin-1, and thrombospondin-2 (TSP-2). We investigated the association between angiogenesis-related factors and clinical outcomes with Nivo. Results: We evaluated 167 samples from 58 pts, including 10 pts (17.2%) exhibiting clinical benefit. Characteristics of pts were as follows: median age, 68; male, 74%; PS, 0/1-2, 17/83%; HER2 positive, 16%; prior gastrectomy, 40%; histology, intestinal/diffuse, 16/84%; disease status, metastatic/recurrent, 69/31%; lung metasstasis, 14%; liver metasstasis, 26%; peritoneum metastasis, 67%; lymph node metastasis, 31%. The median progression-free survival (PFS) and overall survival (OS) of Nivo were 1.7 and 6.2 months, respectively, with a median follow-up of 5.0 months. There were no correlations between pre-Nivo levels of angiogenesis-related factors and clinical benefit; however, the median post-Nivo/pre-Nivo VEGF-A ratio was significantly lower in pts with clinical benefit than in those without clinical benefit (0.44 versus 0.94, p = 0.008). By multivariate analysis using characteristics of pts and data from pre-Nivo samples, VEGF-A above the median level of 1400 pg/mL and sVCAM-1 below the median level of 1640000 pg/mL were independent prognostic factors for poor PFS (hazard ratio, 1.90, p = 0.033) and OS (hazard ratio, 2.02, p = 0.037), respectively. Conclusions: Our study suggests that rapid decline of serum VEGF-A level after Nivo and higher VEGF-A before Nivo were respectively associated with the clinical benefit of Nivo and poor survival in pts with AGC treated with Nivo after RAM-based second-line chemotherapy.

Abstract 14103: Impact of Chronic Kidney Disease on the Association of Cardiovascular and Renal Biomarkers With the Presence and Severity of Coronary Artery Disease in Patients With Suspected Coronary Artery Disease: The EXCEED-J Study

Background: The impact of chronic kidney disease (CKD) on the association of cardiovascular (CV) and renal biomarkers with the presence and severity of coronary artery disease (CAD) in patients with suspected CAD are unclear. Methods: Using data from a multicenter, prospective cohort of 2324 patients with suspected but no history of CAD undergoing elective coronary angiography, we assessed the impact of CKD on the association of CV and renal biomarkers with the presence and severity of CAD. Heparin-free fasting serum levels of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), VEGF, placental growth factor (PlGF), N-terminal pro-brain natriuretic peptide (NT-proBNP), contemporary sensitive cardiac troponin-I (cTnI), high-sensitivity CRP (hs-CRP), cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL) were measured in 887 CKD and 1437 non-CKD patients enrolled in the EXCEED-J study. The severity of CAD was quantified using the Gensini score. Results: The mean age (standard deviation) of the patients was 69.8 (10.8) years; 65.6% were men. Serum levels of all biomarkers except PlGF were significantly higher in CKD than in non-CKD patients. In the entire patient cohort, only natural log-transformed (Ln-) cTnI was significantly associated with CAD, multi-vessel or left main trunk disease (MVD/LMTD), and Ln-Gensini score after adjustment for potential confounders. These associations were still significant in non-CKD, but not in CKD: Ln-cTnI was significantly associated with MVD/LMTD and Ln-Gensini score, but not with CAD. Stepwise regression analyses including potential confounders and all biomarkers revealed that Ln-cTnI was independently associated with CAD, MVD/LMTD, and Ln-Gensini score in the entire cohort and in non-CKD, but not in CKD: Ln-cTnI was independently associated with MVD/LMTD and Ln-Gensini score, but not with CAD. No other biomarker exhibited an independent association with CAD, MVD/LMTD, or Ln-Gensini score in the entire cohort, in non-CKD, or in CKD. Conclusions: Among CV and renal biomarkers measured in this study, only Ln-cTnI was independently associated with the presence and severity of CAD in patients with suspected CAD. These associations were attenuated in patients with CKD.

Abstract 13712: Association Between Soluble Vascular Endothelial Growth Factor Receptor-2 and Prognosis in Patients With Chronic Heart Failure: The PREHOSP-CHF Study

Introduction: Soluble vascular endothelial growth factor receptor-2 (sVEGFR-2) acts as an endogenous inhibitor of VEGF, a key regulator of angiogenesis and lymphoangiogenesis. However, little is known about the critical role of sVEGFR-2 in heart failure (HF). Methods: We performed a multicenter prospective cohort study (PREHOSP-CHF Study) to determine the predictive value of sVEGFR-2 for major adverse cardiovascular (CV) events (MACE) among patients with chronic HF (CHF). A total of 1,021 patients were included in the analyses. The mean age (SD) was 75.5 (12.6) years. 59.6% were male. The primary outcome was MACE defined as a composite of CV death or HF hospitalization. The secondary outcomes were all-cause death and CV death. Serum levels of sVEGFR-2 were determined employing specific enzyme-linked immunosorbent assays. Results: The patients with lower sVEGFR-2 concentrations were older, and had higher rates of female sex, atrial fibrillation and anemia. The baseline sVEGFR-2 level was inversely correlated with left ventricular ejection fraction, and was positively correlated with the body mass index. During the median follow-up of 730 days, a total of 210 (20.6%) all-cause deaths, 99 (9.7%) CV deaths and 308 (30.2%) HF hospitalizations occurred. Unadjusted Cox proportional hazard analyses revealed that the patients with the lowest quartile (Q1) of sVEGFR-2 did not show a significantly higher risk of MACE (p=0.7), but showed the greatest risks of CV death (p=0.003) and all-cause death (p=0.007). Even after adjusting for established risk factors and CV biomarkers (N-terminal pro-brain natriuretic peptide, contemporary sensitive cardiac troponin-I, and high-sensitivity C-reactive protein), those with the Q1 of sVEGFR-2 exhibited the greatest risk of CV death (p=0.02; hazard ratio [HR], 2.05; 95% confidence interval [CI], 1.17-3.66 [vs. Q2]; HR, 2.42; 95%CI, 1.30-4.68 [vs. Q3]; HR, 1.52; 95%CI, 0.80-2.99 [vs. Q4]), but not that of all-cause death. The sex-stratified analyses revealed that the association between sVEGFR-2 and CV death was still significant in men, but not in women (p for interaction, 0.07). Conclusions: A low sVEGFR-2 value was not associated with MACE, but was independently associated with CV mortality among patients with CHF.

Gene polymorphisms and serum levels of sVEGFR-1 in patients with systemic lupus erythematosus

Correlation between soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) concentration, VEGFR1 gene polymorphisms and systemic lupus erythematosus (SLE) risk remains unclear. The present case–control study comprised 254 SLE patients, 385 other rheumatic diseases patients and 390 healthy controls. Serum levels of sVEGFR-1 were detected by enzyme-linked immunosorbent assay. Seven VEGFR1 genetic variants (rs2296188, rs9943922, rs2296283, rs7324510, rs9554322, rs9582036, rs9554320) were genotyped by KASP. Serum levels of sVEGFR-1 were up-regulated in SLE and positively correlated with disease activity. Furthermore, serum sVEGFR-1 presented a distinctive elevation in SLE in comparison with other rheumatic diseases. Frequencies of allele T of rs2296283 and allele G of rs9554322 were significant lower in SLE patients (P = 0.003, P = 0.004). Frequencies of genotypes TT of rs2296188 and rs2296283 were declined in patients compared with healthy controls (P = 0.039, P = 0.033). CC genotype of rs7324510 and rs9582036 was negatively correlated with SLE risk (OR = 0.538, OR = 0.508). Distribution of GG, GC, GG + GC genotypes of rs9554322 were different between SLE patients and healthy controls (P = 0.027, P = 0.036, P = 0.010). Moreover, frequency of TC genotype of rs7324510 was higher in SLE patients with lupus headache (χ2 = 9.924, P = 0.039) and frequency of TC genotype of rs9943922 was lower in patients with cylindruriain (χ2 = 7.589, P = 0.026). Frequencies of allele C of rs7324510 and allele T of rs9943922 were decreased in SLE patients with cylindruria and hypocomplementemia, respectively (χ2 = 4.195, P = 0.041, χ2 = 3.971, P = 0.046). However, frequency of allele C of rs9554322 was increased in SLE patients with pyuria (χ2 = 11.702, P = 0.001). In addition, SLE patients carrying GG, GC, CC genotypes for rs9554322 had higher levels of serum sVEGFR-1. In conclusion, serum sVEGFR-1 was elevated in SLE patients and may be a disease marker. VEGFR1 gene polymorphisms related to risk of SLE in a Chinese Han population.

Abstract 3138: Colorectal cancer is associated with elevated plasma levels of soluble platelet-derived growth factor-dd (PDGF-DD) compared to plasma levels of benign colon disease patients

Abstract Introduction: Platelet-derived growth factor-DD (PDGF-DD), a member of the PDGF family, is secreted as a latent homodimer which is activated by uPA (urokinase). After binding to its receptor, PDGF Rβ, PDGF-DD plays a role in mesenchymal transition, cancer cell growth, migration, and invasion all of which are supported by the PDGF DD induced proangiogenic proteins MMP2, MMP9 and VEGF. Downregulation of PDGF DD suppresses both the expression of VEGF and MMP9 via Notch-1, NF-kappaB and VEGF directed neo-angiogenesis. Upregulated expression of PDGF DD has been noted in colon and many other cancers. This study's purpose was to compare preoperative (PreOp) plasma PDGF DD levels in colorectal cancer (CRC) and benign colon disease (BCD) patients (pts). Method: Pts undergoing colorectal resection for CRC or benign problems that had been prospectively enrolled in an IRB approved tissue and data bank for whom adequate Preop plasma samples were available were studied. Clinical, demographic, operative, and pathologic data were collected. The PDGF-DD levels in the preoperative plasma samples were determined via ELISA in duplicate and reported as median ±95% CI (pg/ml). The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate the CRC diagnostic value of plasma protein levels. The Mann-Whitney test was used for statistical analysis, (significance (sig) p&amp;lt;0.05). Results: A total of 116 CRC (72% colon, 28% rectal) and 86 benign disease pts (adenoma 31%, diverticulitis 55 %, other 14%) were studied. The male: female ratios were similar but the CRC pts were older (p=0.001). The CRC stage distribution was: stage 1, 33 (28%); stage 2, 33 (28%); stage 3, 41 (36%); stage 4, 9 (8%). The median PreOp plasma PDGF DD level was significantly higher in the CRC group (386.8, CI: 341.2, 498.8) than in the benign disease group (320.4, CI: 281.414.5, p&amp;lt;0.001). No correlation was found between PDGF-DD levels and tumor stage. In sub group analysis, tumor expression of PDGF-DD was confirmed by IHC in 8/10 pts (tumor vs. paired normal tissue). The AUC for the ROC curve (AUC) for plasma PDGF DD in association with a diagnosis of CRC was 0.62751 (sensitivity 29%, specificity 95%). Conclusion: Preoperative plasma PDGF-DD levels were evaluated in CRC pts and compared to those in benign colorectal disease pts. The median plasma PDGF-DD level in CRC pts was 21% higher than the median level in BCD pts. AUC value suggests that PDGF-DD may be a potential candidate for a prognostic marker panel for CRC. The higher levels in CRC pts could be attributed to tumor-related inflammation, progression, and angiogenesis. Further studies with a larger population of cancer free control and CRC pts are necessary to better determine whether PDGF-DD levels correlate with Cancer stage and prognosis. Citation Format: Chandana S K Herath Mudiyanselage, Neil Mitra, Dasuni Niyagama Gamage, Hiromichi Miyagaki, Abhinit Shah, Xiaohong Yan, Vesna Cekic, Richard L. Whelan. Colorectal cancer is associated with elevated plasma levels of soluble platelet-derived growth factor-dd (PDGF-DD) compared to plasma levels of benign colon disease patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3138.

Unfractionated heparin displaces sFlt-1 from the placental extracellular matrix

Soluble vascular endothelial growth factor receptor-1 (sFlt-1) is an anti-angiogenic protein which is secreted by numerous cell types and acts as a decoy receptor for the angiogenic protein vascular endothelial growth factor (VEGF). Despite its physiologic importance in maintaining angiogenic balance, excess sFlt-1 levels are associated with the pathogenesis of many diseases, especially those with angiogenic imbalance, endothelial dysfunction, and hypertension. Although sFlt-1 is a soluble protein, it contains a binding site for the extracellular matrix component heparan sulfate. This allows cells to retain and localize sFlt-1 in order to prevent excessive VEGF signaling. During pregnancy, placental syncytiotrophoblasts develop a large extracellular matrix which contains significant amounts of heparan sulfate. Consequently, the placenta becomes a potential storage site for large amounts of sFlt-1 bound to extracellular heparan sulfate. Additionally, it should be noted that sFlt-1 can bind to the anticoagulant unfractionated heparin due to its molecular mimicry to heparan sulfate. However, it remains unknown whether unfractionated heparin can compete with heparan sulfate for binding of localized sFlt-1. In this study, we hypothesized that administration of unfractionated heparin would displace and solubilize placental extracellular matrix(ECM)-bound sFlt-1. If unfractionated heparin can displace this large reservoir of sFlt-1 in the placenta and mobilized it into the maternal circulation, we should be able to observe its effects on maternal angiogenic balance and blood pressure. To test this hypothesis, we utilized in vitro, ex vivo, and in vivo methods. Using the BeWo placental trophoblast cell line, we observed increased sFlt-1 in the media of cells treated with unfractionated heparin compared to controls. The increase in media sFlt-1 was found in conjunction with decreased localized cellular Flt (sFlt-1 and Flt-1) as measured by total cell fluorescence. Similar results were observed using ex vivo placental villous explants treated with unfractionated heparin. Real-time quantitative PCR of the explants showed no change in sFlt-1 or heparanase-1 mRNA expression, eliminating increased production and enzymatic cleavage of heparan sulfate as causes for sFlt-1 media increase. Timed-pregnant rats given a continuous infusion of unfractionated heparin exhibited an increased mean arterial pressure as well as decreased bioavailable VEGF compared to vehicle-treated animals. These data demonstrate that chronic unfractionated heparin treatment is able to displace matrix-bound sFlt-1 into the maternal circulation to such a degree that mean arterial pressure is significantly affected. Here we have shown that the placental ECM is a storage site for large quantities of sFlt-1, and that it should be carefully considered in future studies concerning angiogenic balance in pregnancy.

Assessment of quality and clinical similarity (pharmacokinetic and safety) of HD204, a biosimilar of bevacizumab.

e21556 Background: Prestige Biopharma Pte Ltd is developing HD204, a biosimilar candidate of Bevacizumab (Avastin). Bevacizumab has been approved in the treatment of a variety of metastatic tumours. Bevacizumab, a recombinant humanized monoclonal antibody block angiogenesis which is required for cancer progression by preventing binding of soluble vascular endothelial growth factor (VEGF) to VEGF receptors. Due to heterogeneity nature of antibody therapeutic, the impact on quality of HD204 on safety and pharmacokinetic (PK) was reaffirmed through clinical study to establish clinical similarity between HD204 and Avastin. Methods: Quality attributes identified to influence PK and safety established through comprehensive analytical characterization was used to correlate any potential differences (structural or biological) between the two compounds if any, could result in any clinical meaningful differences in safety and PK in the clinical settings. The PK and safety equivalence of HD204 relative to Avastin was demonstrated in a randomized, single-blind, single-dose, three-arm and parallel-group study clinical Phase I (SAMSON). A total of 120 healthy male subjects randomized 1:1:1 were to receive 1 mg/kg intravenous infusion of either HD204, EU- or US-Avastin. Various PK parameters, safety assessments not limiting to adverse events (AE) and measurement of antidrug antibodies (ADA) and neutralizing antibodies (NAb) were evaluated. Results: The pairwise comparisons of Exposure (AUC0-inf and AUC0-last), maximal concentration (Cmax) established equivalence between the 3 arms. All other PK parameters including half-life, clearance and volume of distribution were comparable between HD204 and Avastin treatment groups. Treatment related TEAEs reported for each group were 25.0%, 30.0% and 25.6% respectively and comparable. There were no treatment-emergent SAEs. Furthermore, none of the subjects treated with HD204 was ADA positive. Conclusions: HD204 demonstrated equivalent PK and safety profile to both US-Avastin and EU-Avastin at 1mg/kg administered as a 90-minute IV infusion to healthy male subjects. A prospective clinical study aimed to demonstrate equivalence in terms of efficacy, PK and safety is currently ongoing. Clinical trial information: 2017-005174-19.

S-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand\u2013foot skin reaction that can be reversed by nicotinamide

Hand–foot skin reaction (HFSR), among the most significant adverse effects of sorafenib, has been limiting the clinical benefits of this frontline drug in treating various malignant tumors. The mechanism underlying such toxicity remains poorly understood, hence the absence of effective intervention strategies. In the present study, we show that vascular endothelial cells are the primary cellular target of sorafenib-induced HFSR wherein soluble heparin-binding epidermal growth factor (s-HBEGF) mediates the crosstalk between vascular endothelial cells and keratinocytes. Mechanistically, s-HBEGF released from vascular endothelial cells activates the epidermal growth factor receptor (EGFR) on keratinocytes and promotes the phosphorylation of c-Jun N-terminal kinase 2 (JNK2), which stabilizes sirtuin 1 (SIRT1), an essential keratinization inducer, and ultimately gives rise to HFSR. The administration of s-HBEGF in vivo could sufficiently induce hyper-keratinization without sorafenib treatment. Furthermore, we report that HBEGF neutralization antibody, Sirt1 knockdown, and a classic SIRT1 inhibitor nicotinamide could all significantly reduce the sorafenib-induced HFSR in the mouse model. It is noteworthy that nicotinic acid, a prodrug of nicotinamide, could substantially reverse the sorafenib-induced HFSR in ten patients in a preliminary clinical study. Collectively, our findings reveal the mechanism of vascular endothelial cell-promoted keratinization in keratinocytes and provide a potentially promising therapeutic strategy for the treatment of sorafenib-induced HFSR.

Soluble neprilysin, NT-proBNP, and growth differentiation factor-15 as biomarkers for heart failure in dialysis patients (SONGBIRD)

BackgroundDialysis patients are at increased risk of HF. However, diagnostic utility of NT-proBNP as a biomarker is decreased in patients on dialysis. GDF-15 and cNEP are biomarkers of distinct mechanisms that may contribute to HF pathophysiology in such cohorts. The aim of this study was to determine whether growth differentiation factor-15 (GDF-15) and circulating neprilysin (cNEP) improve the diagnosis of congestive heart failure (HF) in patients on dialysis.Methods and resultsWe compared circulating concentrations of NT-proBNP, GDF-15, and cNEP along with cNEP activity in patients on chronic dialysis without (n = 80) and with HF (n = 73), as diagnosed by clinical parameters and post-dialysis echocardiography. We used correlation, linear and logistic regression as well as receiver operating characteristic (ROC) analyses. Compared to controls, patients with HF had higher median values of NT-proBNP (16,216 [interquartile range, IQR = 27739] vs. 2883 [5866] pg/mL, p < 0.001), GDF-15 (7512 [7084] vs. 6005 [4892] pg/mL, p = 0.014), but not cNEP (315 [107] vs. 318 [124] pg/mL, p = 0.818). Median cNEP activity was significantly lower in HF vs. controls (0.189 [0.223] vs. 0.257 [0.166] nmol/mL/min, p < 0.001). In ROC analyses, a multi-marker model combining clinical covariates, NT-proBNP, GDF-15, and cNEP activity demonstrated best discrimination of HF from controls (AUC = 0.902, 95% CI 0.857–0.947, p < 0.001 vs. base model AUC = 0.785).ConclusionWe present novel comparative data on physiologically distinct circulating biomarkers for HF in patients on dialysis. cNEP activity but not concentration and GDF-15 provided incremental diagnostic information over clinical covariates and NT-proBNP and may aid in diagnosing HF in dialysis patients.Graphic abstract