VEGF-D Levels Research Articles

The association of cytokine levels and postnatal factors with retinopathy of prematurity.

Prematurity has been known to trigger several cellular pathways, leading to the clinical occurrence of retinopathy of prematurity (ROP). This study compared the levels of a panel of serum cytokines in premature infants with and without ROP. This is a prospective observational study. Premature infants at 36-38 weeks' gestational age were recruited, their clinical data recorded, and serum samples collected and assayed for 18 cytokines. Based on follow-up examinations, patients were divided into two groups: No ROP and ROP. The ROP group was further divided into two subgroups: non-vision-threatening ROP (non-VTROP), and vision-threatening ROP (VTROP). On univariate analysis, among the clinical parameters, gestation age, birth weight, duration of invasive ventilation, and duration of stay in neonatal intensive care unit (NICU) were found to be significant. The univariate analysis also showed an association between raised levels of VEGF-D and IL-8 in the VTROP group. Multiple logistic regression indicated that gestation age was a significant risk factor across all subgroups. Additionally, VEGF-D levels were found to be significantly associated with VTROP. Higher VEGF-D levels are associated with an increased risk of severe ROP that requires treatment and could potentially be used as a biomarker.

Increased Microvascular Filtration and Vascular Endothelial Growth Factor-D associated with Changed Lymphatic Vessel Morphology in Breast Cancer Treated Patients.

Vascular endothelial growth factors (VEGF) and inflammatory cytokines are indicated to be implicated in lymphedema development. We aimed to describe changes in microvascular filtration and VEGFs in a patient cohort vulnerable to breast cancer-related lymphedema development correlated with data on lymphatic morphology and function. Consecutive node-positive breast cancer patients operated in the axilla and evaluated approximately 12 months after adjuvant locoregional radiotherapy were studied. Capillary filtration rate (CFR) and isovolumetric pressure of the arms were measured by strain gauge plethysmography, and 13 blood proteins were quantified by Luminex and Elisa technology in 28 patients and 18 healthy controls. The CFR was reduced in both arms from baseline to 1-year follow-up (ipsilateral: P = 0.016 and contralateral: P = 0.001). When stratifying lymphatic complications (morphologic abnormalities and/or breast cancer-related lymphedema), CFR reached a lower steady-state in the arms with normal morphology (I:P = 0.013 and C:P = 0.013) whereas the ipsilateral arm with lymphatic complications remained unchanged (P = 0.457). In patients with lymphatic abnormal vessels, the levels of VEGF-D were 86% higher than in patients with normal lymphatic vessels (P = 0.042), whereas levels of VEGFR-3 were 64% higher (P = 0.016). Through one year of follow-up, CFR did not decrease in the lymphatic complicated treated arms as observed in noncomplicated treated arms. The patients had increased levels of VEGF-D and VEGFR-3. This correlation suggests that VEGF plays a role in the appearance of subcutaneous abnormal lymphatic vessels in the treated arms, which also maintain a fluid filtration/drainage mismatch up to one year after breast cancer treatment.

Visfatin Facilitates VEGF-D-Induced Lymphangiogenesis through Activating HIF-1α and Suppressing miR-2277-3p in Human Chondrosarcoma.

Chondrosarcoma is a malignant bone tumor that arises from abnormalities in cartilaginous tissue and is associated with lung metastases. Lymphangiogenesis plays an essential role in cancer metastasis. Visfatin is an adipokine reported to enhance tumor metastasis, but its relationship with VEGF-D generation and lymphangiogenesis in chondrosarcoma remains undetermined. Our results from clinical samples reveal that VEGF-D levels are markedly higher in chondrosarcoma patients than in normal individuals. Visfatin stimulation promotes VEGF-D-dependent lymphatic endothelial cell lymphangiogenesis. We also found that visfatin induces VEGF-D production by activating HIF-1α and reducing miR-2277-3p generation through the Raf/MEK/ERK signaling cascade. Importantly, visfatin controls chondrosarcoma-related lymphangiogenesis in vivo. Therefore, visfatin is a promising target in the treatment of chondrosarcoma lymphangiogenesis.

An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer.

CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Causal effect of vascular endothelial growth factor on the risk of atrial fibrillation: a two-sample Mendelian randomization study.

Observational studies have found that vascular endothelial growth factor (VEGF) levels are associated with the risk of cardiovascular disease. However, it remains unclear whether VEGF levels have a causal effect on the risk of atrial fibrillation. A two-sample Mendelian randomization (MR) study was conducted to explore the causal relationship between VEGF levels and the risk of atrial fibrillation. Genetic variants associated with VEGF [VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2 (VEGFR-2), VEGFR-3] and atrial fibrillation (atrial fibrillation, atrial fibrillation and flutter) were used as instrumental variables. Data on genetic variants were obtained from published genome-wide association studies (GWAS) or the IEU Open GWAS project. Inverse-variance weighted (IVW) analysis was used as the primary basis for the results, and sensitivity analyses were used to reduce bias. Causal relationships were expressed as odds ratio (OR) with 95% confidence interval (CI), and a P-value of <0.1 corrected for False Discovery Rate (FDR) (PFDR < 0.1) was considered to have a significant causal relationship. Genetically predicted high levels of VEGF-A [OR = 1.025 (95%CI: 1.004-1.047), PFDR = 0.060] and VEGF-D [OR = 1.080 (95%CI: 1.039-1.123), PFDR = 0.001]] were associated with an increased risk of atrial fibrillation, while no causal relationship was observed between VEGF-C (PFDR = 0.419), VEGFR-2 (PFDR = 0.784), and VEGFR-3 (PFDR = 0.899) and atrial fibrillation risk. Moreover, only genetically predicted high levels of VEGF-D [OR = 1.071 (95%CI: 1.014-1.132), PFDR = 0.087] increased the risk of atrial fibrillation and flutter. Sensitivity analysis demonstrated that the relationship between VEGF-D levels and the risk of atrial fibrillation was robust. This study supports a causal association between high VEGF-D levels and increased risk of atrial fibrillation.

Vascular endothelial growth factor-D plasma levels in fluid overload and cardiac function evaluation of elderly patients with cardiovascular disease.

This study aimed to investigate the clinical significance of vascular endothelial growth factor (VEGF) subtypes and growth differentiation factor-15 (GDF-15) plasma levels in evaluating the fluid overload and cardiac function of elderly patients with cardiovascular disease. The plasma levels of VEGF-C, VEGF-D, and GDF-15 were measured using ELISA. Their correlations with N-terminal pro B-type natriuretic peptide (NT-Pro BNP) and echocardiography data were analyzed. 1. Higher plasma levels of VEGF-D and GDF-15 were observed in elderly patients with cardiovascular disease and heart failure(P < .01). VEGF-D plasma levels were higher in patients with chronic heart failure than those with acute myocardial infarction (P < .01). VEGF-D plasma levels were positively correlated with amino-terminal pro-B type natriuretic peptide (NT-pro BNP) (P < .001). VEGF-D plasma levels were positively correlated with echocardiographic parameters, including left atrial diameter, left ventricular end-diastolic diameter and left ventricular ejection fraction, in patients with cardiovascular disease (P < .01). 2. VEGF-C plasma levels were higher in acute myocardial infarction group (P < .05). The plasma levels of VEGF-C were not correlated with either VEGF-D or NT-pro BNP plasma levels. VEGF-C plasma levels had no correlation with echocardiographic parameters. 3. GDF-15 plasma levels were positively correlated with sera biomarkers of cardiac injury (creatine kinase isoenzyme MB and cardiac troponin I). GDF-15 plasma levels were positively correlated with urinary biomarkers of tubular injury (N-acetyl-β-galactosidase and α1-microglobulin). Both GDF-15 and NT-pro BNP plasma levels were correlated with age, estimated glomerular filtration rate (eGFR), and nutritional biomarkers (albumin and hemoglobin plasma levels). VEGF-D plasma levels is a potential biomarker of fluid overload and cardiac function in elderly patients with cardiovascular disease. Age, nutrition, and kidney injury are factors influencing both GDF-15 and NT-pro BNP plasma levels in estimating cardiac function and fluid overload.

Impact of chronic kidney disease on the association of vascular endothelial growth factor D with cardiovascular mortality in patients with suspected or known coronary artery disease: the ANOX study

Abstract Background Vascular endothelial growth factor D (VEGF-D) is a secreted glycoprotein that can induce lymphangiogenesis and angiogenesis. We recently demonstrated that serum levels of VEGF-D are independently associated with all-cause mortality in patients with suspected or known coronary artery disease (CAD). However, the impact of chronic kidney disease (CKD) on the associations of VEGF-D with cardiovascular (CV) events and mortality in those patients is unclear. Methods Serum VEGF-D levels were measured in 2,418 patients with suspected or known CAD undergoing elective coronary angiography. The primary outcome was CV death. The secondary outcomes were all-cause death, major adverse CV events (MACE) defined as a composite of CV death, nonfatal myocardial infarction, and nonfatal stroke, and heart failure (HF) hospitalization. Patients were divided into 2 groups according to the presence (CKD, n=999) or absence (non-CKD, n=1,419) of CKD, and followed up over a 6-year period. Results During the follow-up, 325 CKD and 211 non-CKD patients died from any cause, 116 CKD and 49 non-CKD patients died from CV disease, 173 CKD and 124 non-CKD patients developed MACE, and 169 CKD and 99 non-CKD patients developed HF hospitalization. After adjustment for potential clinical confounders and established CV biomarkers (i.e., N-terminal pro-brain natriuretic peptide, high-sensitivity cardiac troponin I, and high-sensitivity C-reactive protein), VEGF-D levels were significantly associated with CV death (hazard ratio [HR] for 1-SD increase, 1.20; 95% confidence interval [CI], 1.06–1.37), all-cause death (HR, 1.15; 95% CI, 1.05–1.25), MACE (HR, 1.15; 95% CI, 1.03–1.29), and HF hospitalization (HR, 1.15; 95% CI, 1.02–1.29) in CKD, while VEGF-D levels were significantly associated with HF hospitalization (HR, 1.27; 95% CI, 1.09–1.49), but not with CV death (HR, 0.98; 95% CI, 0.68–1.43), all-cause death (HR, 1.10; 95% CI, 0.94–1.28), or MACE (HR, 0.94; 95% CI, 0.74–1.20) in non-CKD patients. The addition of VEGF-D levels to the model with potential clinical confounders and established CV biomarkers significantly improved the prediction of CV death (P&amp;lt;0.001 for continuous net reclassification improvement [NRI], P= 0.046 for integrated discrimination improvement [IDI]) and all-cause death (P&amp;lt;0.001 for NRI, P=0.004 for IDI), but not that of MACE (P=0.001 for NRI, P= 0.080 for IDI) or HF hospitalization (P=0.542 for NRI, P=0.605 for IDI) in CKD, whereas the addition of VEGF-D levels did not improve the prediction of CV death (P=0.769 for NRI, P=0.717 for IDI), all-cause death (P=0.089 for NRI, P=0.485 for IDI), MACE (P=0.876 for NRI, P=0.313 for IDI) or HF hospitalization (P=0.532 for NRI, P=0.155 for IDI) in non-CKD patients. Conclusions The VEGF-D level independently predicted CV and all-cause mortality in CKD, but not in non-CKD patients with suspected or known CAD. The associations of VEGF-D with CV and all-cause mortality may depend on the presence of CKD.

Evaluation of maternal serum vascular endothelial growth factor C and D levels in intrahepatic cholestasis of pregnancy.

This study aimed to investigate maternal serum vascular endothelial growth factor (VEGF) C and D levels in patients with intrahepatic cholestasis of pregnancy (ICP). A total of 83 patients, including 41 patients with ICP and 42 healthy pregnant women, were included in the study. We first compared the maternal serum VEGF-C and VEGF-D levels between the ICP and control groups and then examined the correlation between the serum VEGF-C level and the bile acid level in patients with severe ICP. We observed statistically significantly higher serum VEGF-C levels and lower VEGF-D levels in the ICP group compared with the healthy controls (P < 0.001 and P = 0.015, respectively). According to receiver operating characteristic analysis, the optimal cutoff value for ICP was 147 ng/mL in the determination of the VEGF-C level (specificity and sensitivity: 76%). In patients with severe ICP, the serum VEGF-C statistically significantly correlated with the bile acid level (P = 0.019). This study showed that the maternal serum VEGF-C level was higher and the VEGF-D level was lower in patients with ICP compared with healthy pregnant women. We also found that the VEGF-C level was correlated with the serum bile acid level in patients with severe ICP. Serum VEGF-C level can be used in the diagnosis and follow-up of intrahepatic pregnancy cholestasis.

Dehydroepiandrosterone supplementation and the impact of follicular fluid metabolome and cytokinome profiles in poor ovarian responders

BackgroundPoor ovarian responders (POR) are women undergoing in-vitro fertilization who respond poorly to ovarian stimulation, resulting in the retrieval of lower number of oocytes, and subsequently lower pregnancy rates. The follicular fluid (FF) provides a crucial microenvironment for the proper development of follicles and oocytes through tightly controlled metabolism and cell signaling. Androgens such as dehydroepiandrosterone (DHEA) have been proposed to alter the POR follicular microenvironment, but the impact DHEA imposes on the FF metabolome and cytokine profiles is unknown. Therefore, the objective of this study is to profile and identify metabolomic changes in the FF with DHEA supplementation in POR patients.MethodsFF samples collected from 52 POR patients who underwent IVF with DHEA supplementation (DHEA +) and without (DHEA-; controls) were analyzed using untargeted liquid chromatography-tandem mass spectrometry (LC–MS/MS) metabolomics and a large-scale multiplex suspension immunoassay covering 65 cytokines, chemokines and growth factors. Multivariate statistical modelling by partial least squares-discriminant regression (PLSR) analysis was performed for revealing metabolome-scale differences. Further, differential metabolite analysis between the two groups was performed by PLSR β-coefficient regression analysis and Student’s t-test.ResultsUntargeted metabolomics identified 118 FF metabolites of diverse chemistries and concentrations which spanned three orders of magnitude. They include metabolic products highly associated with ovarian function – amino acids for regulating pH and osmolarity, lipids such fatty acids and cholesterols for oocyte maturation, and glucocorticoids for ovarian steroidogenesis. Four metabolites, namely, glycerophosphocholine, linoleic acid, progesterone, and valine were significantly lower in DHEA + relative to DHEA- (p < 0.05–0.005). The area under the curves of progesterone glycerophosphocholine, linoleic acid and valine are 0.711, 0.730, 0.785 and 0.818 (p < 0.05–0.01). In DHEA + patients, progesterone positively correlated with IGF-1 (Pearson r: 0.6757, p < 0.01); glycerophosphocholine negatively correlated with AMH (Pearson r: -0.5815; p < 0.05); linoleic acid correlated with estradiol and IGF-1 (Pearson r: 0.7016 and 0.8203, respectively; p < 0.01 for both).In DHEA- patients, valine negatively correlated with serum-free testosterone (Pearson r: -0.8774; p < 0.0001). Using the large-scale immunoassay of 45 cytokines, we observed significantly lower MCP1, IFNγ, LIF and VEGF-D levels in DHEA + relative to DHEA.ConclusionsIn POR patients, DHEA supplementation altered the FF metabolome and cytokine profile. The identified four FF metabolites that significantly changed with DHEA may provide information for titrating and monitoring individual DHEA supplementation.

Lymphangioleiomyomatosis: circulating levels of FGF23 and pulmonary diffusion.

Lymphangioleiomyomatosis (LAM) is a rare, destructive disease of the lungs with a limited number of determinants of disease activity, which are a critical need for clinical trials. FGF23 has been implicated in several chronic pulmonary diseases. We aimed to determine the association between serum FGF23 levels and pulmonary function in a cohort of patients with LAM. This was a descriptive single-center study in which subjects with LAM and controls with unreported lung disease were recruited. Serum FGF23 levels were measured in all subjects. Clinical data, including pulmonary function testing, were retrospectively obtained from electronic medical records of LAM subjects. Associations between FGF23 levels and clinical features of LAM were explored via nonparametric hypothesis testing. The sample comprised 37 subjects with LAM and 16 controls. FGF23 levels were higher in the LAM group than in the control group. In the LAM group, FGF23 levels above the optimal cutoff point distinguished 33% of the subjects who had nondiagnostic VEGF-D levels. Lower FGF23 levels were associated with impaired DLCO (p = 0.04), particularly for those with isolated diffusion impairment with no other spirometric abnormalities (p = 0.04). Our results suggest that FGF23 is associated with pulmonary diffusion abnormalities in LAM patients and elicit novel mechanisms of LAM pathogenesis. FGF23 alone or in combination with other molecules needs to be validated as a biomarker of LAM activity in future clinical research.

Associations Between CSF Markers of Inflammation, White Matter Lesions, and Cognitive Decline in Individuals Without Dementia.

Small vessel disease (SVD) and neuroinflammation both occur in Alzheimer disease (AD) and other neurodegenerative diseases. It is unclear whether these processes are related or independent mechanisms in AD, especially in the early stages of disease. We therefore investigated the association between white matter lesions (WML; the most common manifestation of SVD) and CSF biomarkers of neuroinflammation and their effects on cognition in a population without dementia. Individuals without dementia from the Swedish BioFINDER study were included. The CSF was analyzed for proinflammatory markers (interleukin [IL]-6 and IL-8), cytokines (IL-7, IL-15, and IL-16), chemokines (interferon γ-induced protein 10, monocyte chemoattractant protein 1), markers of vascular injury (soluble intercellular adhesion molecule 1, soluble vascular adhesion molecule 1), and markers of angiogenesis (placental growth factor [PlGF], soluble fms-related tyrosine kinase 1 [sFlt-1], vascular endothelial growth factors [VEGF-A and VEFG-D]), and amyloid β (Aβ)42 Aβ40, and p-tau217. WML volumes were determined at baseline and longitudinally over 6 years. Cognition was measured at baseline and follow-up over 8 years. Linear regression models were used to test associations. A total of 495 cognitively unimpaired (CU) elderly individuals and 247 patients with mild cognitive impairment (MCI) were included. There was significant worsening in cognition over time, measured by Mini-Mental State Examination, Clinical Dementia Rating, and modified preclinical Alzheimer composite score in CU individuals and patients with MCI, with more rapid worsening in MCI for all cognitive tests. At baseline, higher levels of PlGF (β = 0.156, p < 0.001), lower levels of sFlt-1 (β = -0.086, p = 0.003), and higher levels of IL-8 (β = 0.07, p = 0.030) were associated with more WML in CU individuals. In those with MCI, higher levels of PlGF (β = 0.172, p = 0.001), IL-16 (β = 0.125, p = 0.001), IL-8 (β = 0.096, p = 0.013), IL-6 (β = 0.088, p = 0.023), VEGF-A (β = 0.068, p = 0.028), and VEGF-D (β = 0.082, p = 0.028) were associated with more WML. PlGF was the only biomarker that was associated with WML independent of Aβ status and cognitive impairment. Longitudinal analyses of cognition showed independent effects of CSF inflammatory markers and WML on longitudinal cognition, especially in people without cognitive impairment at baseline. Most neuroinflammatory CSF biomarkers were associated with WML in individuals without dementia. Our findings especially highlight a role for PlGF, which was associated with WML independent of Aβ status and cognitive impairment.

Vascular endothelial growth factor-D plasma levels and VEGFD genetic variants are independently associated with outcomes in patients with cardiovascular disease.

The vascular endothelial growth factor (VEGF) family is involved in pathophysiological mechanisms underlying cardiovascular (CV) diseases. The aim of this study was to investigate the associations between circulating VEGF ligands and/or soluble receptors and CV outcome in patients with acute coronary syndrome (ACS) and chronic coronary syndrome (CCS). Levels of VEGF biomarkers, including bFGF, Flt-1, KDR (VEGFR2), PlGF, Tie-2, VEGF-A, VEGF-C, and VEGF-D, were measured in the PLATO ACS cohort (n = 2091, discovery cohort). Subsequently, VEGF-D was also measured in the STABILITY CCS cohort (n = 4015, confirmation cohort) to verify associations with CV outcomes. Associations between plasma VEGF-D and outcomes were analysed by multiple Cox regression models with hazard ratios (HR [95% CI]) comparing the upper vs. the lower quartile of VEGF-D. Genome-wide association study (GWAS) of VEGF-D in PLATO identified SNPs that were used as genetic instruments in Mendelian randomization (MR) meta-analyses vs. clinical endpoints. GWAS and MR were performed in patients with ACS from PLATO (n = 10 013) and FRISC-II (n = 2952), and with CCS from the STABILITY trial (n = 10 786). VEGF-D, KDR, Flt-1, and PlGF showed significant association with CV outcomes. VEGF-D was most strongly associated with CV death (P = 3.73e-05, HR 1.892 [1.419, 2.522]). Genome-wide significant associations with VEGF-D levels were identified at the VEGFD locus on chromosome Xp22. MR analyses of the combined top ranked SNPs (GWAS P-values; rs192812042, P = 5.82e-20; rs234500, P = 1.97e-14) demonstrated a significant effect on CV mortality [P = 0.0257, HR 1.81 (1.07, 3.04) per increase of one unit in log VEGF-D]. This is the first large-scale cohort study to demonstrate that both VEGF-D plasma levels and VEGFD genetic variants are independently associated with CV outcomes in patients with ACS and CCS. Measurements of VEGF-D levels and/or VEGFD genetic variants may provide incremental prognostic information in patients with ACS and CCS.

Human gingival epithelial cells stimulate proliferation, migration, and tube formation of lymphatic endothelial cells invitro.

The aim of this study was to investigate the response of gingival epithelial cells to microbial and inflammatory signals. The gingival epithelial barrier provides the first line of defense and supports tissue homeostasis by maintaining the cross-talk between gingival epithelium, oral microbiota, and immune cells. Lymphatic vessels are essential to sustaining this homeostasis. The gingival epithelial cells have been shown to produce prolymphangiogenic factors during physiologic conditions, but their role in response to microbial and inflammatory signals is unknown. Immortalized human gingival epithelial cells (HGEC) and human dermal lymphatic microvascular endothelial cells (LEC) were cultured. HGEC were exposed to Porphyromonas gingivalis derived-LPS, human IL-1 beta/IL-1F2 protein, or recombinant human IL-6/IL-6R. Levels of vascular growth factors (VEGF-A, VEGF-C, and VEGF-D) in cell supernatants were determined by ELISA. LEC were grown to confluence, and a scratch was induced in the monolayer. Uncovered area was measured up to 48 h after exposure to conditioned medium (CM) from HGEC. Tube formation assays were performed with LEC cocultured with labelled HGEC or exposed to CM. VEGF-A, VEGF-C, and low levels of VEGF-D were constitutively expressed by HGEC. The expression of VEGF-C and VEGF-D, but not VEGF-A, was upregulated in response to proinflammatory mediators. VEGF-C was upregulated in response to P.gingivalis LPS, but not to Escherichia coli LPS. A scratch migration assay showed that LEC migration was significantly increased by CM from HGEC. Both the CM and coculture with HGEC induced significant tube formation of LEC. HGEC can regulate production of lymphangiogenic/angiogenic factors during inflammatory insults and can stimulate proliferation, migration, and tube formation of LEC in vitro in a paracrine manner.

Changes in Serum Growth Factors during Resistance to Atezolizumab Plus Bevacizumab Treatment in Patients with Unresectable Hepatocellular Carcinoma.

The possible mechanisms of resistance to atezolizumab/bevacizumab for unresectable HCC, and the subsequent response to these therapies, remain underexplored. The sequential changes in serum growth factors, including VEGF-A, VEGF-C, VEGF-D, ANG-2, FGF-19, HGF, and EGF during atezolizumab/bevacizumab for unresectable HCC were evaluated in 46 patients. Patients who experienced PD after CR, PR, or SD to atezolizumab/bevacizumab were evaluated. A total of 4, 9, 19, and 14 patients showed CR, PR, SD, and PD, respectively. Of 32 patients with disease control, 28 experienced PD after CR, PR, or SD with atezolizumab/bevacizumab. Baseline growth factor levels were similar between patients with or without disease control and those with or without an objective response. Growth factor changes between the baseline and the best overall response points (BOR) for patients with disease control showed that FGF-19 significantly increased and ANG2 significantly decreased at the BOR. Growth factor changes between the BOR and the PD point in 28 patients who experienced PD after disease control showed that VEGF-D and ANG2 significantly increased at the PD point compared with that at the BOR. Summarily, increased serum VEGF-D and ANG-2 levels might contribute to developing resistance to atezolizumab/bevacizumab for unresectable HCC and might be target molecules in subsequent salvage therapies.

Magrolimab, Rituximab and Acalabrutinib for Relapsed/Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL): Results from the Phase 1 PRISM Trial

Background: R/R DLBCL patients (pts) who fail multiple lines of treatment or are ineligible for novel treatments, including CAR T-cell therapy, have poor clinical outcomes. The combination of the anti-CD47 monoclonal antibody (mAb) magrolimab and rituximab enhances macrophage-mediated antibody-dependent cellular phagocytosis in DLBCL (Advani et al. NEJM 2019). We hypothesized that the Bruton's tyrosine kinase inhibitor acalabrutinib would further enhance clinical activity in R/R DLBCL. We present the final analysis of Arm 3 of the Phase 1 PRISM platform study of acalabrutinib with magrolimab and rituximab in all molecular subtypes of R/R DLBCL (NCT03527147). Methods:Adults with DLBCL and ECOG PS ≤2 who had failed ≥1 line of prior chemoimmunotherapy or were ineligible for high-dose chemotherapy with stem-cell rescue and/or CAR T-cell therapy were eligible. Pts with prior CAR T-cell therapy were excluded from the dose-limiting toxicity (DLT) evaluable pts. Pts received magrolimab 1 mg/kg IV priming dose on cycle 1 day 1 (C1D1) of a 28-day cycle, then 30 mg/kg IV on D8, D15, and D22, on D1, D8, D15, and D22 of C2, and on D1 and D15 thereafter. Rituximab 375 mg/m2 was given on D8, D15, and D22 of C1, D1 of C2-6 and every other cycle from C8 onwards. Acalabrutinib was given continuously at 100 mg twice daily. Combination treatment was given until disease progression or for a maximum of 2 years. DLTs were evaluated from first treatment administration until the end of C1. The primary objective was safety of the combination regimen. Secondary objectives included response rate, pharmacokinetics (PK), and immunogenicity. Exploratory analyses included baseline tumor samples, baseline and longitudinal immunophenotyping of peripheral blood, and cytokine and mutation analysis of plasma samples. Results:By 31 March 2021, 7 pts with DLBCL (including 3 with transformed disease) were enrolled. Median age was 72 years (51-84). Pts had received a median of 4 prior regimens (range 1-6). Three (42.9%) pts were refractory to their most recent regimen. No DLTs were observed in 6 DLT-evaluable pts. Five pts discontinued treatment due to progressive disease (PD), 1 due to an adverse event (AE), and 1 due to study termination. Grade ≥3 AEs regardless of causality reported in ≥10% of pts included anemia (42.9%), thrombocytopenia (28.6%), bilirubin elevation (14.3%), vomiting (14.3%), and ALT elevation (14.3%). Two pts experienced serious AEs (1 pneumonitis, 1 septic shock). Overall, the safety profile was consistent with magrolimab monotherapy studies. No treatment-related deaths were reported. Two (28.6%) pts had an objective response per Response Evaluation Criteria in Lymphoma (RECIL) 2017; both were complete responses (CR). At a median follow-up of 8.3 months, response durations for these pts were 0.03 and 7.36 months. One CR pt had a non-germinal center B-cell (GCB) DLBCL; the other was unknown. Of the 2 PD pts, 1 had non-GCB DLBCL with a BN2/A53 genetic subtype and 1 had GCB DLBCL with an EZB/A53 genetic subtype. Fluctuations in cell-free DNA (cfDNA) and cytokines tended to correlate with response by imaging. Both PD pts had higher plasma cfDNA concentrations than other pts (1 pt at screening, C1D1, and C1D8 and the other at C1D15, C2D1, and end of treatment). CR pts had lower mutational burden and lower variant allele frequencies than PD pts at screening and longitudinally. The allele frequencies of copy number alterations in tissue samples at screening were also higher in stable disease and PD pts. PD pts had higher baseline levels of ICAM1, IL-10, IL-15, IL-6, MCP-1, MIP1a, MIP1b, TNF-b, VEGF-A, and VEGF-D than non-PD pts. PK data were consistent with monotherapy results and no antibodies to magrolimab were detected. Conclusions:Acalabrutinib combined with magrolimab and rituximab was safe and tolerable in pts with advanced R/R DLBCL and no prior exposure to CAR T-cell therapy. The safety profile of the combination was consistent with the known safety profiles of the individual agents. However, due to the lack of obvious clinical synergy of the triplet over acalabrutinib monotherapy or the combination of magrolimab and rituximab, the study was stopped prematurely. Conclusions from cfDNA and cytokine analyses are limited by small pt numbers, but such analyses should be included in future studies of targeted combinations because mounting evidence suggests they tend to correlate with response.

Acute-phase Serum Cytokine Levels and Correlation with Clinical Outcomes in Children and Adults with Primary and Secondary Dengue Virus Infection in Myanmar between 2017 and 2019.

The dengue virus (DENV) has been endemic in Myanmar since 1970, causing outbreaks every 2–3 years. DENV infection symptoms range from mild fever to lethal hemorrhage. Clinical biomarkers must be identified to facilitate patient risk stratification in the early stages of infection. We analyzed 45 cytokines and other factors in serum samples from the acute phase of DENV infection (within 3–5 days of symptom onset) from 167 patients in Yangon, Myanmar, between 2017 and 2019. All of the patients tested positive for serum DENV nonstructural protein 1 antigen (NS1 Ag); 78.4% and 62.9% were positive for immunoglobulin M (IgM) and G (IgG), respectively; and 18.0%, 19.8%, and 11.9% tested positive for serotypes 1, 3, and 4, respectively. Although the DENV-4 viral load was significantly higher than those of DENV-1 or DENV-3, disease severity was not associated with viral load or serotype. Significant correlations were identified between disease severity and CCL5, SCF, PDGF-BB, IL-10, and TNF-α levels; between NS1 Ag and SCF, CCL5, IFN-α, IL-1α, and IL-22 levels; between thrombocytopenia and IL-2, TNF-α, VEGF-D, and IL-6 levels; and between primary or secondary infection and IL-2, IL-6, IL-31, IL-12p70, and MIP-1β levels. These circulating factors may represent leading signatures in acute DENV infections, reflecting the clinical outcomes in the dengue endemic region, Myanmar.

Abstract 105: Proprotein Convertase Subtilisin/kexin Type 6 (Pcsk6) Plays A Role In Angiogenesis Regulation

Background: PCSK6 cleaves and activates growth factors involved in cell differentiation and Pcsk6-/- mice exhibit 25% embryonic lethality, while surviving offspring have blindness and cyclopia. We have shown that PCSK6 localizes to vascular smooth muscle cells of intra-plaque neovessels. We therefore hypothesize that the processes of angiogenesis, atherosclerosis and plaque neovascularisation share common molecular regulators and that PCSK6 plays a role in these processes. Methods: Association between SNPs near the PCSK6 gene and levels of 90 plasma proteins was tested using data from 30,931 individuals in the SCALLOP consortium. Associations of angiogenic factors with PCSK6 (transcriptomic and proteomic) and patient symptomatology were explored in a biobank of plaque endarterectomies (BiKE). Morpholino technology was used for Pcsk6 knockdown in zebrafish, coupled with vascular phenotype studies. Pcsk6-/- mice were used to study the expression of vasculogenesis related genes and vascular morphology at baseline (12 weeks). Results: Rs7178801 and rs11639051 in the PCSK6 gene were found to relate to plasma PDGFB and VEGFD levels. TGFB2, VEGFA, VEGFC, PDGFA and PLAUR associated with patients symptomatology in BiKE and correlated with PCSK6 levels. Gross phenotypic and histological examination of zebrafish embryos with ablated PCSK6 showed improper peripheral vascular patterning of intersegmental vessels along with cerebral and myocardial haemorrhage hemorrhage. Expression of angiogenic factors; Vegfa, Vegfb, Angpt1 and Tgfb2 were altered in the heart, liver and adipose tissue in Pcsk6-/- mice. Vessel density, length and branch numbers were reduced in the Pcsk6 -/- compared to the controls in the superficial but more significantly in the deep peripheral vessels. The smooth muscle cell coverage of the retinal vasculature was also reduced in the Pcsk6 -/- mice vs controls and Pdgfa was significantly downregulated compared to controls in mouse retinas. Conclusions: PCSK6 associates with levels of angiogenic factors on the genetic, transcriptomic and proteomic level. Lack of Pcsk6 in mice and zebrafish led to vascular patterning defects. Further planned studies will examine PCSK6 in pathological neovascularization.

Serum VEGF-D level is correlated with renal dysfunction and proteinuria in patients with diabetic chronic kidney disease.

Biomarkers associated with chronic kidney disease (CKD) may play a crucial role in the early diagnosis of diabetic kidney disease. However, there have been few reports published on serum vascular endothelial cell growth factor (VEGF)-D in patients with diabetic CKD. We divided patients with diabetic CKD into two groups: CKD 3–4 and CKD 5. In total, 42 patients with diabetic kidney disease and seven healthy controls without diabetes mellitus were enrolled in this study. An observational study was conducted to evaluate the serum VEGF-D levels and other clinical parameters in each group and to assess the relationship among these factors. The serum levels of VEGF-D were higher in the CKD 3–4 group and CKD 5 group than in the control group. However, there was no significant difference in serum levels of VEGF-D between CKD stage 3–4 group and CKD stage 5 group. Correlation analysis showed that serum VEGF-D was negatively correlated with estimated glomerular filtration rate but positively correlated with serum creatinine, urine albumin-to-creatinine ratio, and urine protein-to-creatinine ratio. Serum VEGF-D was a good biomarker in receiver operating characteristic analysis and independently associated with CKD stages in multiple linear regression analysis. Circulating VEGF-D was positively correlated with blood growth/differentiation factor-15, endostatin, and chemokine (C-X-C motif) ligand 16 levels. Serum VEGF-D levels were correlated with renal dysfunction, albuminuria, and proteinuria in patients with diabetic kidney disease. Elucidation of the role of VEGF-D as a biomarker requires further study.

Dysregulated Serum Lipid Metabolism Promotes the Occurrence and Development of Diabetic Retinopathy Associated With Upregulated Circulating Levels of VEGF-A, VEGF-D, and PlGF.

Purpose: This study aims to explore the correlations of arteriosclerosis-associated plasma indices with various severity levels of diabetic retinopathy (DR) and to test the hypothesis that elevated circulating level of known angiogenic cytokines induced by hyperglycemia is associated with dyslipidemia on DR.Methods: This cross-sectional study consists of 131 patients with type 2 diabetes. The patients were categorized based on their DR status into those with no DR (diabetes mellitus, DM), non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR) groups. The biochemical profile including fasting glucose, glycated hemoglobin (HbA1c), lipid profile were estimated, plasma angiogenic cytokines (vascular endothelial growth factor, VEGF-A, -C, -D) and placental growth factor (PlGF) were analyzed by protein microarrays. The atherogenic plasma index (API) was defined as low-density lipoprotein cholesterol/high-density lipoprotein cholesterol (LDL-C/HDL-C); atherogenic index (AI) was calculated as (TC-(HDL-C))/HDL-C and atherogenic index of plasma (AIP) was defined as log (TG/HDL-C).Results: No significant differences were detected in the duration of hypertension, age, and gender between the three groups. Serum TC and LDL-C, AI, and API in the NPDR group and PDR group were significantly higher than those in the DM group. The circulating level of PlGF, VEGF-A, and VEGF-C were significantly correlated with the severity of DR. VEGF-D is a risk factor independent of API (Z = −2.61, P = 0.009) and AI (Z = −2.40, P = 0.016). Multivariate logistic regression showed that AI and API are strong risk factors for the occurrence and severity of DR. Associated with AI and API, VEGF-D and PlGF contribute to DR: VEGF-D [AI: P = 0.038, odd ratio (OR) = 1.38; VEGF-D: P = 0.002, OR = 1.00. API: P = 0.027, OR = 1.56, VEGF-D:P = 0.002, OR = 1.00] and PlGF [AI: P = 0.021, OR = 1.43; VEGF-D: P = 0.004, OR = 1.50. API: P = 0.011, OR = 1.66; VEGF-D: P = 0.005, OR = 1.49].Conclusions: Total cholesterol (TC) and LDL-C are risk factors for presence of any DR. Atherogenic index and API are novel and better predictive indicators for the occurrence and severity of DR in comparion with the traditional lipid profiles. Abnormal lipid metabolism are associated with the upregulation of circulating cytokines that are linked to the severity of DR.

Changes in Blood Biomarkers of Angiogenesis and Immune Modulation after Radiation Therapy and Their Association with Outcomes in Thoracic Malignancies.

Simple SummaryRadiation therapy can promote chemotaxis of cytotoxic T-lymphocytes by triggering the release of chemokines and altering the tumor’s vascular endothelium, triggering both pro- and anti-inflammatory immune responses and altering the tumor microenvironment. These effects of local irradiation may have systemic consequences and can be enhanced through the combination of available immune checkpoint blockers (ICBs). The study and validation of minimally invasive blood biomarkers for response and toxicity assessment are critical to stratify patients that would benefit from combination treatments. This exploratory prospective study evaluated the impact of thoracic radiotherapy approaches on the immune system using longitudinal assessment of a panel of blood biomarkers of angiogenesis and inflammation. We show that changes in circulating TNF-α, IL-6 and IL-8 levels could potentially indicate an early reduction in immunosuppression after radiotherapy. If validated in larger studies, these biomarker candidates might potentially help in optimally scheduling radiotherapy in combination with ICBs.The effects of radiotherapy on systemic immunity remain to be fully characterized in a disease-specific manner. The aim of the study was to examine potential biomarkers of systemic immunomodulation when using radiotherapy for thoracic malignancies. Serial blood samples were collected from 56 patients with thoracic malignancies prior (RTbaseline), during (RTduring) and at the end of radiotherapy (RTend), as well as at the first (FU1) and second follow-up (FU2). The changes in serum levels of IL-10, IFN-γ, IL-12p70, IL-13, IL-1β, IL-4, IL-6, IL-8, TNF-α, bFGF, sFlt-1, PlGF, VEGF, VEGF-C, VEGF-D and HGF were measured by multiplexed array and tested for associations with clinical outcomes. We observed an increase in the levels of IL-10, IFN-γ, PlGF and VEGF-D and a decrease in those of IL-8, VEGF, VEGF-C and sFlt-1 during and at the end of radiotherapy. Furthermore, baseline concentration of TNF-α significantly correlated with OS. IL-6 level at RTend and FU1,2 correlated with OS (RTend: p = 0.039, HR: 1.041, 95% CI: 1.002–1.082, FU1: p = 0.001, HR: 1.139, 95% CI: 1.056–1.228, FU2: p = 0.017, HR: 1.101 95% CI: 1.018–1.192), while IL-8 level correlated with OS at RTduring and RTend (RTduring: p = 0.017, HR: 1.014, 95% CI: 1.002–1.026, RTend: p = 0.004, HR: 1.007, 95% CI: 1.061–1.686). In conclusion, serum levels of TNF-α, IL-6 and IL-8 are potential biomarkers of response to radiotherapy. Given the recent implementation of immunotherapy in lung and esophageal cancer, these putative blood biomarkers should be further validated and evaluated in the combination or sequential therapy setting.