Expression Of PGF Research Articles

A novel cancer-associated fibroblast signature for kidney renal clear cell carcinoma via integrated analysis of single-cell and bulk RNA-sequencing

Cancer-associated fibroblasts (CAFs), integral components of the tumor microenvironment, play a pivotal role in tumor proliferation, metastasis, and clinical outcomes. However, its specific roles in Kidney Renal Clear Cell Carcinoma (KIRC) remain poorly understood. Employing the established Seurat single-cell analysis pipeline, we identified 21 CAFs marker genes. Subsequently, a prognostic signature consisting of 6 CAFs marker genes (RGS5, PGF, TPM2, GJA4, SEPT4, and PLXDC1) was developed in a cohort through univariate and LASSO Cox regression analyses. The model’s efficacy was then validated in an external cohort, with a remarkable predictive performance in 1-, 3-, and 5-year. Patients in the high-risk group exhibited significantly inferior survival outcomes (p < 0.001), and the risk score was an independent prognostic factor (p < 0.05). Distinct differences in immune cell profiles and drug susceptibility were observed between the two risk groups. In KIRC, the PGF-VEGFR1 signaling pathway displayed a notable increase. PGF expression was significantly elevated in tumor tissues, as demonstrated by quantitative real-time polymerase chain reaction. In vitro, transwell assays and CCK8 revealed that recombinant-PGF could enhance the capability of cell proliferation, migration, and invasion in 769P and 786-O cells. This study firstly developed a novel predictive model based on 6 CAFs genes for KIRC. Additionally, PGF may present a potential therapeutic target to enhance KIRC treatment.

Fructose Consumption Affects Placental Production of H2S: Impact on Preeclampsia-Related Parameters.

H2S, a gasotransmitter that can be produced both via the transsulfuration pathway and non-enzymatically, plays a key role in vasodilation and angiogenesis during pregnancy. In fact, the involvement of H2S production on plasma levels of sFLT1, PGF, and other molecules related to preeclampsia has been demonstrated. Interestingly, we have found that maternal fructose intake (a common component of the Western diet) affects tissular H2S production. However, its consumption is allowed during pregnancy. Thus, (1) to study whether maternal fructose intake affects placental production of H2S in the offspring, when pregnant; and (2) to study if fructose consumption during pregnancy can increase the risk of preeclampsia, pregnant rats from fructose-fed mothers (10% w/v) subjected (FF) or not (FC) to a fructose supplementation were studied and compared to pregnant control rats (CC). Placental gene expression, H2S production, plasma sFLT1, and PGF were determined. Descendants of fructose-fed mothers (FC) presented an increase in H2S production. However, if they consumed fructose during their own gestation (FF), this effect was reversed so that the increase disappeared. Curiously, placental synthesis of H2S was mainly non-enzymatic. Related to this, placental expression of Cys dioxygenase, an enzyme involved in Cys catabolism (a molecule required for non-enzymatic H2S synthesis), was significantly decreased in FC rats. Related to preeclampsia, gene expression of sFLT1 (a molecule with antiangiogenic properties) was augmented in both FF and FC dams, although these differences were not reflected in their plasma levels. Furthermore, placental expression of PGF (a molecule with angiogenic properties) was decreased in both FC and FF dams, becoming significantly diminished in plasma of FC versus control dams. Both fructose consumption and maternal fructose intake induce changes in molecules that contribute to increasing the risk of preeclampsia, and these effects are not always mediated by changes in H2S production.

Integrating Mendelian randomization and single-cell RNA sequencing to identify therapeutic targets of baicalin for type 2 diabetes mellitus.

Alternative and complementary therapies play an imperative role in the clinical management of Type 2 diabetes mellitus (T2DM), and exploring and utilizing natural products from a genetic perspective may yield novel insights into the mechanisms and interventions of the disorder. To identify the therapeutic target of baicalin for T2DM, we conducted a Mendelian randomization study. Druggable targets of baicalin were obtained by integrating multiple databases, and target-associated cis-expression quantitative trait loci (cis-eQTL) originated from the eQTLGen consortium. Summary statistics for T2DM were derived from two independent genome-wide association studies available through the DIAGRAM Consortium (74,124 cases vs. 824,006 controls) and the FinnGen R9 repository (9,978 cases vs. 12,348 controls). Network construction and enrichment analysis were applied to the therapeutic targets of baicalin. Colocalization analysis was utilized to assess the potential for the therapeutic targets and T2DM to share causative genetic variations. Molecular docking was performed to validate the potency of baicalin. Single-cell RNA sequencing was employed to seek evidence of therapeutic targets' involvement in islet function. Eight baicalin-related targets proved to be significant in the discovery and validation cohorts. Genetic evidence indicated the expression of ANPEP, BECN1, HNF1A, and ST6GAL1 increased the risk of T2DM, and the expression of PGF, RXRA, SREBF1, and USP7 decreased the risk of T2DM. In particular, SREBF1 has significant interaction properties with other therapeutic targets and is supported by strong colocalization. Baicalin had favorable combination activity with eight therapeutic targets. The expression patterns of the therapeutic targets were characterized in cellular clusters of pancreatic tissues that exhibited a pseudo-temporal dependence on islet cell formation and development. This study identified eight potential targets of baicalin for treating T2DM from a genetic perspective, contributing an innovative analytical framework for the development of natural products. We have offered fresh insights into the connections between therapeutic targets and islet cells. Further, fundamental experiments and clinical research are warranted to delve deeper into the molecular mechanisms of T2DM.

Effects of a combination of cannabidiol and delta-9-tetrahydrocannabinol on key biological functions of HTR-8/SVneo extravillous trophoblast cells

In recent years, cannabis use has increased among pregnant women. In addition, the phytocannabinoids cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) alone or in combination are being used for therapeutical applications. THC and CBD are able to cross the placenta and a lot remains unknown concerning their impact on angiogenesis and extravillous trophoblasts’ (EVTs) migration and invasion, which are essential processes for placentation. Thus, in this study, the HTR-8/SVneo cell line was employed to evaluate the effects of CBD, THC and of their combination (1:1, 2 µM). Cannabinoids affected epithelial-mesenchymal transition, as showed by increased expression of the epithelial protein marker E-cadherin for CBD and CBD plus THC treatments, and decrease of mesenchymal intermediate filament vimentin for all treatments. The gene expression of the metalloproteinases MMP2 and MMP9, and of their inhibitors TIMP1 and TIMP2 was increased, except the latter for THC treatment. Moreover, CBD reduced cell migration and invasion, an effect that was enhanced by its combination with THC. CBD with or without THC also upregulated the gene expression of PGF, while the anti-angiogenic factor sFLT1 was increased for all treatments. VEGFA and FLT1 were not affected. Alone or combined CBD and THC also decreased tube segments’ length. Additionally, ERK1/2 and STAT3 phosphorylation was increased in the CBD and CBD plus THC-treated cells, while THC only activated STAT3. AKT activation was only affected by CBD. This work demonstrates that the exposure to cannabinoid-based products containing CBD and/or THC, may interfere with key processes of EVTs differentiation. Therefore, crucial phases of placental development can be affected, compromising pregnancy success.

Role of stemness-related genes TIMP1, PGF, and SNAI1 in the prognosis of colorectal cancer through single-cell RNA-seq.

Colorectal cancer (CRC) is a fatal malignant tumor with poor prognosis. Cancer stem cells (CSCs) can cause metastasis, recurrence and drug resistance in CRC. This research aimed to analyze stemness-related prognostic genes of CRC based on single-cell RNA-sequencing (scRNA-seq) data. DESeq2 was applied to analyze the differentially expressed genes (DEGs). The mRNA stemness index (mRNAsi) was calculated by one-class logistic regression (OCLR). The stemness-related cells were analyzed based on scRNA-seq dataset GSE166555. Monocle 2 algorithm was used for stemness-related cells pseudotime trajectory analysis. The stemness-related prognostic genes were analyzed by clusterProfiler and survival package. The stemness of CRC cells was detected by spheroid formation assay, and the expression of stemness-related prognostic genes was verified by qRT-PCR and Western blot. 7916 DEGs between the CRC and normal tissues were obtained. The mRNAsi of the CRC tissues was shown to be significantly higher than that of the normal tissues. 7 and 8 cell types were annotated respectively in the normal and CRC tissues through analysis of the scRNA-seq data. Cell-cell interactions (CCIs) in the tumor tissues were revealed to be significantly enhanced than that in the normal tissues. By calculating the 'stemness score', CSCs, epithelial cells (EPCs) and cancer-associated fibroblasts (CAFs) were defined as stemness-related cells. Through pseudotime trajectory analysis, 2111 genes were identified as state 2-specific genes. Then, 41 genes were obtained by taking intersection of the up-regulated genes with state 2-specific genes and marker genes of CSCs, EPCs and CAFs. The univariate COX regression analysis revealed 5 stemness-related prognostic genes (TIMP1, PGF, FSTL3, SNAI1 and FOXC1). Kaplan-Meier curve analysis indicated that the higher the expression of 5 genes, the lower the survival rate. In vitro cell experiment confirmed that the expression of TIMP1, PGF and SNAI1 was consistent with that revealed by bioinformatics analysis. TIMP1, PGF and SNAI1 were identified as stemness-related prognostic genes of CRC, and possibly potential therapeutic targets for CRC.

Placental DAAM2 is unaltered in preeclampsia, but upregulated by treatment with proton pump inhibitors.

Dishevelled Associated Activator Of Morphogenesis 2 (DAAM2) levels are elevated in the maternal circulation and placenta in pregnancies complicated by fetal growth restriction. However, placental DAAM2 levels in cases of preeclampsia have not previously been explored. Here, we examined placental DAAM2 in pregnancies complicated by preterm preeclampsia, and whether candidate preeclampsia therapeutics altered its expression. DAAM2 mRNA and protein levels were assessed in placental tissue from cases of preterm preeclampsia and gestation-matched controls (delivering ≤ 34weeks; qPCR and western blot respectively). Short interfering RNAs were used to silence DAAM2 in isolated primary cytotrophoblast under normoxic (8% O2) and hypoxic (1% O2) conditions, and expression of anti-angiogenic sFLT-1, angiogenic PGF, antioxidant, fetal growth, and inflammatory genes assessed. DAAM2 expression was measured in placental explant tissue from pregnancies complicated by preeclampsia, treated with three proton pump inhibitors (100µM esomeprazole, lansoprazole, and rabeprazole). DAAM2 expression was significantly reduced in preeclamptic placental tissue compared to controls, but protein production was unchanged. Silencing DAAM2 in hypoxic cytotrophoblast increased sFLT-i13 isoform expression, but did not alter sFLT-e15a or PGF expression, or sFLT-1 secretion. DAAM2 knockdown did not alter expression of antioxidant (NQO-1, TXN, GCLC), fetal growth (SPINT1), or inflammasome (NLRP3) genes. Esomeprazole and lansoprazole, but not rabeprazole, increased DAAM2 expression in placental explant tissue from cases of preeclampsia. Placental DAAM2 protein is not significantly altered in placental tissue in cases of preeclampsia, and its suppression does not alter sFLT-1 secretion. Hence, placental DAAM2 is unlikely to drive the pathogenesis associated with preeclampsia.

Whole Blood Expression of the Vascular Endothelial Growth Factor Family Relates to Cognitive Performance

AbstractBackgroundThe vascular endothelial growth factor (VEGF) family is involved in angiogenic signaling and has been implicated in Alzheimer’s Disease (AD). Previous work from our team has found that higher transcript levels of VEGFB, PGF, FLT1, and FLT4 in the brain relate to worse cognitive performance and higher levels of AD neuropathology. This study explored whether comparable associations are observed when measuring mRNA in whole blood.MethodTranscriptomic, cognitive, and biomarker data were acquired from the Vanderbilt Memory &amp; Aging Project. Participants (n=335) included older adults (age=72+/‐7.32) with normal cognition (n=203) or mild cognitive impairment (n=132). Expression of the ten genes in the VEGF family were quantified using RNA sequencing of blood. Outcomes included a composite measure of memory and cerebrospinal fluid (CSF) levels of β‐amyloid, total tau, and phosphorylated tau (ptau181). Linear regression models related VEGF family member expression to baseline cognitive performance and AD biomarkers. Linear mixed effects regression models assessed longitudinal change in cognition. All models covaried for age at baseline and sex. To aid in interpretation, Pearson correlations between blood and brain expression were calculated leveraging data from 137 participants from the Genotype Tissue Expression Project (GTexportal.org).ResultAt baseline, higher levels of VEGFB mRNA were associated with better memory performance (Figure 1, β=0.47, p=0.008). In longitudinal analyses, higher baseline expression of PGF was associated with more rapid decline in memory (β= 0.02, p=0.04). No other significant associations were observed (p‐values&gt;0.09). Among GTEx participants, higher expression of VEGFB in blood was correlated with lower expression in the brain (Figure 2, r=‐0.19, p=0.02). PGF correlations did not reach statistical significance (r=0.09, p=0.27).ConclusionHigher PGF expression in blood relates to worse cognitive outcomes, consistent with previous findings in the brain. In contrast, higher levels of VEGFB in blood relates to better performance, opposite of the previously observed association in the brain. Notably, in GTEx we observed opposing correlations between blood levels of VEGFB and brain levels of VEGFB, suggesting that higher levels of VEGFB in the blood may indeed reflect lower levels in the brain. Our results highlight the potential of VEGFB and PGF as blood biomarkers in aging and AD.

Targeting TRAF3IP2 inhibits angiogenesis in glioblastoma.

Increased vascularization, also known as neoangiogenesis, plays a major role in many cancers, including glioblastoma multiforme (GBM), by contributing to their aggressive growth and metastasis. Although anti-angiogenic therapies provide some clinical improvement, they fail to significantly improve the overall survival of GBM patients. Since various pro-angiogenic mediators drive GBM, we hypothesized that identifying targetable genes that broadly inhibit multiple pro-angiogenic mediators will significantly promote favorable outcomes. Here, we identified TRAF3IP2 (TRAF3-interacting protein 2) as a critical regulator of angiogenesis in GBM. We demonstrated that knockdown of TRAF3IP2 in an intracranial model of GBM significantly reduces vascularization. Targeting TRAF3IP2 significantly downregulated VEGF, IL6, ANGPT2, IL8, FZGF2, PGF, IL1β, EGF, PDGFRB, and VEGFR2 expression in residual tumors. Our data also indicate that exogenous addition of VEGF partially restores angiogenesis by TRAF3IP2-silenced cells, suggesting that TRAF3IP2 promotes angiogenesis through VEGF- and non-VEGF-dependent mechanisms. These results indicate the anti-angiogenic and anti-tumorigenic potential of targeting TRAF3IP2 in GBM, a deadly cancer with limited treatment options.

Combination of sunitinib and 177Lu-labeled antibody cG250 targeted radioimmunotherapy: A promising new therapeutic strategy for patients with advanced renal cell cancer

Sunitinib is an effective treatment for patients with metastatic Renal Cell Carcinoma (mRCC) but ultimately resistance occurs. The aim of this study was to investigate sunitinib resistance in RCCs and to develop therapeutic combination strategies with targeted radioimmunotherapy (RIT).We studied two RCC models, analyzed Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) and AXL/MET expression and performed therapy studies in Balb/cnu/nu mice combining sunitinib and [177Lu]Lu-cG250 RIT (6.5 MBq/10 μg), specifically targeting RCC cells.pAXL and pMET were expressed in sunitinib-resistant SK-RC-52 and absent in sunitinib-sensitive NU12. NGS evaluation showed that expression of VEGFA, VEGFB, VEGFD, PGF and VEGFR1,2,3 was higher and expression of VEGFC and PDGFA was lower in NU12 than in SK-RC-52.Therapy studies combining sunitinib with [177Lu]Lu-cG250 RIT showed that the best response in mice with “resistant” SK-RC-52 tumors was observed with two cycles of Sunitinib and [177Lu]Lu-cG250 RIT, probably due to increased vascular permeability by sunitinib treatment. In the “sensitive” NU12 model, two cycles of [177Lu]Lu-cG250 RIT and two cycles of combination treatment were equally effective.Enhanced therapeutic efficacy was achieved when two agents ([177Lu]Lu-cG250 RIT and sunitinib) that on their own did not induce satisfactory response levels, are combined. Our findings provide a promising new therapeutic strategy for patients with advanced RCC.

Sprouting Angiogenesis in Human Pituitary Adenomas.

IntroductionAngiogenesis in pituitary tumors is not fully understood, and a better understanding could help inform new pharmacologic therapies, particularly for aggressive pituitary tumors.Materials and Methods219 human pituitary tumors and 12 normal pituitary glands were studied. Angiogenic genes were quantified by an angiogenesis qPCR array and a TaqMan probe-based absolute qPCR. Angiogenesis inhibition in pituitary tumors was evaluated in vitro with the endothelial tube formation assay and in vivo in RbΔ19 mice.Results71 angiogenic genes, 40 of which are known to be involved in sprouting angiogenesis, were differentially expressed in pituitary tumors. Expression of endothelial markers CD31, CD34, and ENG was significantly higher in pituitary tumors, by 5.6, 22.3, and 8.2-fold, respectively, compared to in normal pituitary tissue. There was no significant difference in levels of the lymphatic endothelial marker LYVE1 in pituitary tumors compared with normal pituitary gland tissue. Pituitary tumors also expressed significantly higher levels of angiogenesis growth factors, including VEGFA (4.2-fold), VEGFB (2.2), VEGFC (19.3), PGF (13.4), ANGPT2 (9.2), PDGFA (2.7), PDGFB (10.5) and TGFB1 (3.8) compared to normal pituitary tissue. Expression of VEGFC and PGF was highly correlated with the expression of endothelial markers in tumor samples, including CD31, CD34, and ENG (endoglin, a co-receptor for TGFβ). Furthermore, VEGFR inhibitors inhibited angiogenesis induced by human pituitary tumors and prolonged survival of RbΔ19 mice.ConclusionHuman pituitary tumors are characterized by more active angiogenesis than normal pituitary gland tissue in a manner consistent with sprouting angiogenesis. Angiogenesis in pituitary tumors is regulated mainly by PGF and VEGFC, not VEGFA and VEGFB. Angiogenesis inhibitors, such as the VEGFR2 inhibitor cabozantinib, may merit further investigation as therapies for aggressive human pituitary tumors.

Inhibitory effect on subretinal fibrosis by anti-placental growth factor treatment in a laser-induced choroidal neovascularization model in mice.

To investigate whether anti-placental growth factor (PGF) can inhibit subretinal fibrosis and whether this effect is mediated by the inhibitory effect of PGF on epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Subretinal fibrosis model was established in laser induced choroidal neovascularization (CNV) mice on day 21 after laser photocoagulation. Immunofluorescence staining (IFS) of cryosections and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of PGF. IFS of whole choroidal flat-mounts was used to detect the degree of subretinal fibrosis. IFS of cryosections and ELISA were used to detect the expression of EMT related indicators in subretinal fibrosis lesions. The expression of PGF protein in subretinal fibrosis lesions was significantly up-regulated (P<0.05), and mainly co-stained with pan-cytokeratin labeled RPE cells. Intravitreal injection of anti-PGF neutralizing antibody reduced the area of subretinal fibrosis and the ratio of fibrotic/angiogenic area significantly at the concentrations of 0.25, 0.5, 1.0, and 2.0 µg/µL (all P<0.05). The expression of E-cadherin in the local RPE cells decreased, while α-SMA increased significantly in subretinal fibrosis lesions, and the application of anti-PGF neutralizing antibody could reverse these changes (P<0.05). The expression of PGF is up-regulated in the lesion site of subretinal fibrosis and mainly expressed in RPE cells. Intravitreal injection of anti-PGF neutralizing antibody can significantly inhibit the degree of subretinal fibrosis in CNV mice, and this effect may be mediated by the inhibition of PGF on EMT of RPE cells.

Gestational exposure to BDE-209 induces placental injury via the endoplasmic reticulum stress-mediated PERK/ATF4/CHOP signaling pathway

Several epidemiological studies have reported significant associations between prenatal polybrominated diphenyl ethers (PBDEs) exposure and adverse birth outcomes. Placental injury is thought to mediate these associations. However, few study has investigated the adverse effects of PBDEs exposure on placental growth and development. We examined the impacts of gestational exposure to BDE-209, the most abundant PBDE conger detected in human samples, on placental structure and function, and its model of action in vivo and in vitro. Pregnant mice were exposed to 0, 2, 20, 200 mg/kg/day of BDE-209 by gavages from gestational day (GD) 0 to GD18. Results showed that gestational BDE-209 exposure significantly reduced placental weight, impaired placental vascular development and induced placental cell apoptosis. In addition, gestational BDE-209 exposure impaired placental transport and endocrine function as demonstrated by markedly downregulated expression of Glut1, Znt1, Pgf and Igf2 in BDE-209-treated placentas. Mechanistically, gestational exposure to BDE-209 upregulated the expression of GRP78, and 3 downstream proteins (p-eIF2α, ATF4 and CHOP) of the PERK signaling, suggesting the activation of endoplasmic reticulum (ER) stress and PERK signaling pathway in mouse placentas. Further in vitro study showed that PERK siRNA pretreatment markedly reversed BDE-209-induced cell apoptosis in human JEG-3 cells. Collectively, our results suggest that the activation of the ER stress-mediated PERK/ATF4/CHOP signaling pathway played a role in BDE-209-induced placental injury. Our findings provide new insight into the mechanisms of BDE-209 induced reproductive and developmental toxicity.

VEGF-family brain protein abundance: Associations with Alzheimer's disease pathology and cognitive decline.

Previous work has implicated associations between the angiogenic vascular endothelial growth factor (VEGF) gene family and Alzheimer's disease (AD) and associated cognitive decline. Specifically, we have shown that mRNA expression of VEGFB, PGF, FLT1, and FLT4 are associated with AD diagnosis, lower cognition prior to death, faster cognitive decline, and higher AD neuropathology at death. However, we wanted to explore whether these associations persisted at the protein level. Detailed cognitive and neuropathological data were obtained from 1,084 participants in the Religious Orders Study and Rush Memory and Aging Project. Selected reaction monitoring mass spectrometry was used to quantify VEGF protein levels in dorsolateral prefrontal cortex tissue. A total of 22 peptides representing five VEGF family proteins were available for analysis. Linear regression assessed VEGF proteomic associations with cognition adjacent to death and with amyloid and tau burden at autopsy. Mixed-effects regression quantified VEGF associations with cognitive decline. Models covaried for age at death, sex, post-mortem interval, and interval to death, with secondary models covarying for post-mortem amyloid and tau burden. P-values were corrected using the false discovery rate (fdr) procedure. Higher levels of VEGFB and FLT1 peptides were associated with lower cognition prior to death (p.fdr≤0.031), as well as with faster cognitive decline (p.fdr≤0.016), while NRP1 peptides were associated with slower cognitive decline (p.fdr=0.010). VEGFB, VEGFA, and FLT1 peptides were positively associated with higher amyloid burden (p.fdr≤0.012), while NRP1 peptides were negatively associated (p.fdr=0.003). VEGFB and FLT1 peptides were also associated with higher tau burden at death (p.fdr≤0.041). Cognitive associations remained when covarying for neuropathology burden. Finally, only FLT1 peptides were differently expressed in AD cases and controls (β≥0.12, p.fdr≤0.024). Proteomic results are highly consistent with previous mRNA findings, strengthening the hypothesis that VEGF's role in AD-related cognitive decline centers on VEGFB-FLT1 signaling. The associations remained largely significant even when covarying for pathological burden, suggesting VEGF family signaling independently contributes to the clinical manifestation of AD. Interestingly, since NRP1 amplifies VEGF family angiogenic signaling, NRP1's association with slower cognitive decline at the protein level may reflect a partially-successful attempt to rescue cognition by increasing blood flow.

Abstract 404: A novel 4-gene score predict patient survival as well as pathologically complete (R0) resection in pancreatic cancer

Abstract BACKGROUND: Pancreatic cancer is one of the most aggressive cancers with a 5-year survival of less than 10%. Surgery is the only cure for pancreatic cancer but the majority of patients present with metastatic disease at the time of diagnosis. Systemic chemotherapy remains the primary treatment option. Given the insidious clinical presentation and poor treatment response, there is an urgent need to discover a predictive biomarker for appropriate patient selection. We hypothesized that the 4-gene score, which reflects tumor cell proliferation, is both a prognostic and predictive biomarker for pancreatic cancer. METHODS: The 4-gene score is derived from tumor expression of DOK4, HCCS, PGF, and SHCBP1 genes, which were identified based on differential mRNA expression analysis of a human breast cell line, its metastatic variant cells, and clinical outcome data of breast cancer patient cohorts, as previously reported. A total of 954 pancreatic cancer patients were analyzed for both discovery and validation of the 4-gene score from publicly available datasets to investigate the relationship between the score with clinical features such as metastasis, cancer aggressiveness, immune cell infiltration, patient survival, and resectability. RESULTS: We found that the 4-gene score correlation in pancreatic cancer was higher than in breast cancer cohorts, specifically in clinically aggressive parameters such as pathological grade and MKI67 expression. Also, the score in metastatic tumor cohorts was higher than in primary cancer cohorts (p &amp;lt; 0.001). These findings were mirrored in gene set analysis, in which the score high tumors enriched cell proliferation-related gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, mitotic spindle, and metastasis-related gene sets in three cohorts (TCGA, GSE62452, and GSE57495). A high score was associated with increased mutation rates in KRAS and CDKN2A genes (p = 0.006 and 0.044) in the TCGA cohort, as well as enhancement of KRAS, CDKN2A, p53, TGF-β, and E2F pathways in three cohorts. Furthermore, a high score tumor had less infiltration of CD8+ T cells but was associated with high cytolytic activity and IFN-γ scores, which reflect overall cancer immunity. The high score was correlated with the level of drug sensitivity area under the curve of Sorafenib (r = 0.648) in primary cancer, Paclitaxel (r = 0.624) and Doxorubicin (r = 0.575) in metastatic cancer. Finally, a high 4-gene score was significantly associated with worse clinical outcomes in three cohorts (p = 0.044, 0.048, and 0.022), a low rate of R0 resection (p = 0.013), and worse survival after R1/2 resection (p = 0.027). CONCLUSION: The 4-gene score identified poor survival in pancreatic cancer and has potential as a predictive biomarker for R0 resection and treatment response in metastatic pancreatic cancer. Evaluating the degree of the 4-gene score could be a valuable potential prognostic tool. Citation Format: Masanori Oshi, Lan Le, Yoshihisa Tokumaru, Ankit Patel, Ryusei Matsuyama, Itaru Endo, Li Yan, Matthew H.G. Katz, Kazuaki Takabe. A novel 4-gene score predict patient survival as well as pathologically complete (R0) resection in pancreatic cancer [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 404.

Decidualization modulates the mesenchymal stromal/stem cell and pericyte characteristics of human decidual stromal cells. Effects on antigen expression, chemotactic activity on monocytes and antitumoral activity

Decidual stromal cells (DSCs) are the most abundant cellular component of human decidua and play a central role in maternal–fetal immune tolerance. Antigen phenotyping and functional studies recently confirmed the relationship of DSCs with mesenchymal stem/stromal cells (MSCs) and pericytes, the latter two cell types being closely related or identical. The present study investigated the effect of decidualization, a process of cell differentiation driven by progesterone (P4) and other pregnancy hormones, on the MSC/pericyte characteristics of DSCs. To this end we isolated undifferentiated DSC (preDSC) lines that were decidualized in vitro (dDSC) by the effect of P4 and cAMP. Using flow cytometry, we found significant downmodulation of the expression of the MSC/pericyte markers α-smooth muscle actin, nestin, CD140b, CD146 and SUSD2 in dDSCs. The dDSCs did not differ, compared to preDSCs, in the expression of angiogenic factors (characteristic of pericytes) HGF, FGF2, ANGPT1 or VEGF according to RT-PCR results, but had significantly increased PGF expression. In migration assays, preDSC-conditioned media had a chemotactic effect on the THP-1 monocytic line (characteristic of pericytes), and this effect was significantly greater in dDSC-conditioned media. Media conditioned with dDSC, but not with preDSC, induced apoptosis in 4 out of 6 different tumor cell lines (characteristic of MSCs) according to propidium iodide staining and flow cytometry results. Our findings show that decidualization induces phenotypic and functional changes in the MSC/pericyte properties of DSCs that may have a role in the normal development of pregnancy.

Hypoxia Induces Galectin-1 Expression Via Autoinduction of Placental Growth Factor in Retinal Pigment Epithelium Cells.

PurposeGalectin-1/LGALS1, a β-galactoside-binding protein, contributes to angiogenesis and fibrosis in various ocular diseases. Hypoxia-dependent and -independent pathways upregulate galectin-1/LGALS1 expression in Müller glial cells. Here, we present novel findings on the galectin-1/LGALS1 regulatory system in human retinal pigment epithelium (RPE) cells, the major cellular participant in the pathogenesis of neovascular age-related macular degeneration (nAMD).MethodsHuman RPE cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and immunoblot analyses, respectively. The promoter and enhancer regions of LGALS1 were analyzed by reporter assay and chromatin immunoprecipitation. Immunofluorescence analysis of nAMD patient specimens was used to confirm the in vitro findings.ResultsHypoxia induced galectin-1/LGALS1 expression via binding of hypoxia-inducible factor 1α (HIF-1α) to hypoxia-responsive elements in the LGALS1 promoter region. Blockade of vascular endothelial growth factor receptor 1 (VEGFR1) partially decreased hypoxia-induced galectin-1/LGALS1 expression. Among several VEGFR1 ligands induced by hypoxia, placental growth factor (PlGF)/PGF alone upregulated galectin-1/LGALS1 expression via phosphorylation of activator protein 1 (AP-1) subunits following AKT and p38 mitogen-activated protein kinase (MAPK) activation. An AP-1 site in the LGALS1 enhancer region was required for PlGF-induced galectin-1/LGALS1 expression in RPE cells. PlGF application upregulated PGF expression via extracellular signal-regulated kinase 1 and 2, AKT, and p38 MAPK pathways. nAMD patient specimens demonstrated co-localization of galectin-1 with HIF-1α, PlGF, and VEGFR1 in RPE cells.ConclusionsOur present findings implicate the significance of hypoxia as a key inducer of galectin-1/LGALS1 in RPE cells and the autoinduction of hypoxia-induced PlGF as a vicious cycle amplifying the pathogenesis of nAMD.

Involvement of Hdac3-mediated inhibition of microRNA cluster 17-92 in bronchopulmonary dysplasia development

BackgroundThe incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, has been paradoxically rising despite medical advances. Histone deacetylase 3 (Hdac3) has been reported to be a crucial regulator in alveologenesis. Hence, this study aims to investigate the mechanism of Hdac3 in the abnormal pulmonary angiogenesis and alveolarization of BPD.MethodsA hyperoxia-induced BPD model of was developed in newborn mice, and primary lung fibroblasts were isolated from adult mice. Hdac3 was knocked out in vivo and knocked down in vitro, while microRNA (miR)-17 was downregulated in vivo and in vitro to clarify their roles in abnormal pulmonary angiogenesis and alveolarization. Mechanistic investigations were performed on the interplay of Hdac3, miR-17-92 cluster, enhancer of zeste homolog 1 (EZH1), p65 and placental growth factor (Pgf).ResultsHdac3 was involved in abnormal alveolarization and angiogenesis in BPD mice. Further, the expression of the miR-17-92 cluster in BPD mice was downregulated by Hdac3. miR-17 was found to target EZH1, and Hdac3 rescued the inhibited EZH1 expression by miR-17 in lung fibroblasts. Additionally, EZH1 augmented Pgf expression by recruiting p65 thus enhancing the progression of BPD. Hdac3 augmented the recruitment of p65 in the Pgf promoter region through the miR-17/EZH1 axis, thus enhancing the transcription and expression of Pgf, which elicited abnormal angiogenesis and alveolarization of BPD mice.ConclusionsAltogether, the present study revealed that Hdac3 activated the EZH1-p65-Pgf axis through inhibiting miR-17 in the miR-17-92 cluster, leading to accelerated abnormal pulmonary angiogenesis and alveolarization of BPD mice.

Abstract 6712: Analysis of VEGF as a potential complimentary target with immunotherapy

Abstract Background: Angiogenesis is a hallmark of malignancy and greatly contributes to tumor growth and dissemination to metastatic sites. Vascular endothelial growth factors (VEGFs) are major members of angiogenesis signaling and have been shown to have immunosuppressive function. Bevacizumab (Bev), a VEGF inhibitor, in combination with immune checkpoint inhibitor (ICI) therapy and chemotherapy, has been shown in a previous clinical trial to improve outcomes in lung cancer versus chemotherapy plus Bev, even in PDL1-low and negative tumors. (IMPOWER150). Tumor mutation burden (TMB) has been established as a biomarker of ICI response, independent of PDL1. We set out to explore the relationship between VEGF signaling and TMB. Methods: We performed a retrospective analysis of data from the NantHealth database of 2,737 commercial cases where matched WGS or WES and RNA-Seq were performed. Primary and metastatic breast tumors were included in the analysis. TMB was computed as the count of SNVs per tumor sample. Bowtie2, RSEM, and custom software are used for alignment, transcript quantification and variant analysis. Per-tumor type analysis was performed to find VEGF signaling correlates with TMB and a panel of immune markers (n = 9). Results: Overall TMB does not correlate with PDL1, PDL2, CTLA4 and other immune markers' expression (spearman range [-0.01 - 0.15]). We also observed that overall TMB does not exhibit strong correlations with VEGF signaling markers (spearman range [-.17 - .17]), however when limited to TMB-low tumors VEGFA and VEGFD positively correlate with TMB (p = 1e-5 and 4.1e-2) and VEGFE negatively correlates with TMB (p = 1.6e-4). We then limited our analysis to PDL1-low tumors, of which the top three categories by sample counts are breast (n = 103), colon (n = 85), and sarcoma (n = 78). In whole-cohort analysis, we observed significantly higher expression of VEGFE and VEGFR2 (p = 1.4e-2 and 2.8e-2) in TMB-low tumors. When performing the same analysis in individual tissue types we observed significantly differentially higher expression of VEGFA and PGF (p = 2.1e-2 and 3.3e-2) in TMB-high colon samples, of VEGFR2 (p = 1.5e-2) in TMB-low NSCLC tumors, and of VEGFR2, FOS, and FOXO1(p = 1.9e-2, 3.4e-2, 6.2e-3) in TMB-low uterine endometrial tumors. Conclusions: We provide evidence that targeting VEGF signaling in some tumor types, especially in tumors with low TMB, might provide a boost to therapeutic effects of checkpoint inhibitors. Our findings warrant further translational interrogation of the potential efficacy with a combination of VEGF inhibitors and ICI in various cancer types. For example, NCT03971474, a trial of ramucirumab (anti-VEGFR2 MAb) plus pembrolizumab in NSCLC, may produce differential activity in TMB-High v TMB-Low cohorts. Citation Format: Yulia Newton, Christopher W. Szeto, Stephen C. Benz, Sandeep K. Reddy. Analysis of VEGF as a potential complimentary target with immunotherapy [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 6712.

Brain expression of the vascular endothelial growth factor gene family in cognitive aging and alzheimer\u2019s disease

Vascular endothelial growth factor (VEGF) is associated with the clinical manifestation of Alzheimer’s disease (AD). However, the role of the VEGF gene family in neuroprotection is complex due to the number of biological pathways they regulate. This study explored associations between brain expression of VEGF genes with cognitive performance and AD pathology. Genetic, cognitive, and neuropathology data were acquired from the Religious Orders Study and Rush Memory and Aging Project. Expression of ten VEGF ligand and receptor genes was quantified using RNA sequencing of prefrontal cortex tissue. Global cognitive composite scores were calculated from 17 neuropsychological tests. β-amyloid and tau burden were measured at autopsy. Participants (n = 531) included individuals with normal cognition (n = 180), mild cognitive impairment (n = 148), or AD dementia (n = 203). Mean age at death was 89 years and 37% were male. Higher prefrontal cortex expression of VEGFB, FLT4, FLT1, and PGF was associated with worse cognitive trajectories (p ≤ 0.01). Increased expression of VEGFB and FLT4 was also associated with lower cognition scores at the last visit before death (p ≤ 0.01). VEGFB, FLT4, and FLT1 were upregulated among AD dementia compared with normal cognition participants (p ≤ 0.03). All four genes associated with cognition related to elevated β-amyloid (p ≤ 0.01) and/or tau burden (p ≤ 0.03). VEGF ligand and receptor genes, specifically genes relevant to FLT4 and FLT1 receptor signaling, are associated with cognition, longitudinal cognitive decline, and AD neuropathology. Future work should confirm these observations at the protein level to better understand how changes in VEGF transcription and translation relate to neurodegenerative disease.

Placental expression of angiogenesis-related genes and their receptors in IUGR pregnancies: correlation with fetoplacental and maternal parameters

Objectives: Aberrations in placental vascular development compromising fetal supply of oxygen and essential nutrients can be a significant contributor to intrauterine growth restriction (IUGR). The development of placental vascular tree is under the influence of two families of growth factors, namely the vascular endothelial growth factor (VEGF) family and angiopoietin/TEK family. In this study, we have examined the expression of angiogenesis-related growth factors, mainly VEGF family and angiopoietin-TEK (endothelial-specific receptor tyrosine kinase) family genes in placentae from IUGR pregnancies uncomplicated by preeclampsia (PE) compared to normal pregnancies.Methods: Placentae from normotensive IUGR (n = 42) and appropriate for gestational age (AGA) pregnancies (n = 47) were collected and examined histologically. Clinical parameters were obtained from the medical records. Real-time quantitative PCR was performed to assess placental transcript abundance of VEGF, PGF, FLT1, ANGPT1, ANGPT2, and TEK normalized to a panel of reference genes. Associations of placental transcript abundance of the genes with maternal, placental, and neonatal parameters were tested.Results: Placental transcript abundance for VEGF (relative expression 10.81 versus 12.98, p < .001), PGF (12.14 versus 13.8, p < .001) and ANGPT2 (3.67 versus 9.55, p = .002) were significantly lower in IUGR placentae compared to AGA. The transcript level of VEGF showed significant negative correlation with birth weight (r = −0.419, p = .006), placental weight (r = −0.318, p = .040), placental length (r = −0.389, p = .011) and breadth (r = −0.308, p = .047) only in the IUGR group. Presence of histopathological features of hypoxia correlated with significantly higher transcript levels of PGF in IUGR placentae (12.6 versus 10.9, p = .046).Conclusion: The low levels of VEGF transcripts may be responsible for the impaired angiogenesis in IUGR placentae. The significance of higher relative expression of PGF in the presence of chronic hypoxia needs to be explored.