BACKGROUND

Clinical factors predicting pulmonary complications after lung resection have been well described, whereas the role of genetics is unknown. The vascular endothelial growth factor (VEGF) signaling pathway has been linked to acute lung injury. We hypothesized that genetic variations in this pathway may be associated with postoperative pulmonary complications after lung resection.

METHODS

One hundred ninety-six single nucleotide polymorphisms (SNPs) in 17 genes in the VEGF pathway were genotyped in a discovery set of 264 patients and a replication set of 264 patients who underwent lobectomy for lung cancer. Multivariable analysis adjusting for baseline clinical factors was used to identify SNPs associated with pulmonary complications. Cumulative and classification and regression tree (CART) analyses were used to further stratify risk groups.

RESULTS

The overall number of pulmonary complications was 164/528 (31%). The effects of 6 SNPs were consistent in the discovery and replication sets (pooled p value<0.05). The rs9319425 SNP in the VEGF receptor gene FLT1 resulted in a 1.50-fold increased risk (1.15-1.96; p=0.003). A cumulative effect for the number of risk genotypes and complications was also evident (p<0.01). Patients carrying 5 risk genotypes had a 5.76-fold increase in risk (2.73-12.16; p=4.44×10(-6)). Regression tree analysis identified potential gene-gene interactions between FLT1:rs9319425 and RAF1:rs713178. The addition of the 6 SNPs to the clinical model increased the area under the receiver operating characteristic curve by 6.8%.

CONCLUSIONS

Genetic variations in the VEGF pathway are associated with risk of pulmonary complications after lobectomy. This may offer insight into the underlying biological mechanisms of pulmonary complications.

BACKGROUND

The FUS-DDIT3 fusion oncogene encodes an abnormal transcription factor that has a causative role in the development of myxoid/round-cell liposarcomas (MLS/RCLS). We have previously identified FLT1 (VEGFR1) as a candidate downstream target gene of FUS-DDIT3. The aim of this study was to investigate expression of FLT1 and its ligands in MLS cells.

METHODS

HT1080 human fibrosarcoma cells were transiently transfected with FUS-DDIT3-GFP variant constructs and FLT1 expression was measured by quantitative real-time PCR. In addition, FLT1, PGF, VEGFA and VEGFB expression was measured in MLS/RCLS cell lines, MLS/RCLS tumors and in normal adiopocytes. We analyzed nine cases of MLS/RCLS and one cell line xenografted in mice for FLT1 protein expression using immunohistochemistry. MLS/RCLS cell lines were also analyzed for FLT1 by immunofluorescence and western blot. MLS/RCLS cell lines were additionally treated with FLT1 tyrosine kinase inhibitors and assayed for alterations in proliferation rate.

RESULTS

FLT1 expression was dramatically increased in transfected cells stably expressing FUS-DDIT3 and present at high levels in cell lines derived from MLS. The FLT1 protein showed a strong nuclear expression in cells of MLS tissue as well as in cultured MLS cells, which was confirmed by cellular fractionation. Tissue array analysis showed a nuclear expression of the FLT1 protein also in several other tumor and normal cell types including normal adipocytes. The FLT1 ligand coding gene PGF was highly expressed in cultured MLS cells compared to normal adipocytes while the other ligand genes VEGFA and VEGFB were expressed to lower levels. A more heterogeneous expression pattern of these genes were observed in tumor samples. No changes in proliferation rate of MLS cells were detected at concentrations for which the kinase inhibitors have shown specific inhibition of FLT1.

CONCLUSIONS

Our results imply that FLT1 is induced as an indirect downstream effect of FUS-DDIT3 expression in MLS. This could be a consequence of the ability of FUS-DDIT3 to hijack parts of normal adipose tissue development and reprogram primary cells to a liposarcoma-like phenotype. The findings of nuclear FLT1 protein and expression of corresponding ligands in MLS and normal tissues may have implications for tissue homeostasis and tumor development through auto- or intracrine signaling.

Phosphatase and tensin homolog (PTEN) acts as a novel tumor suppressor gene. PTEN protein plays an important role in regulating proliferation, apoptosis, invasion, and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating PI3K/Akt pathway in many solid tumors. However, the effects of PTEN on VEGF and VEGFR1 (FLT1)-mediated angiogenesis, migration, invasion of leukemia cells, and its clinical significance are still unknown in myeloid leukemia. Therefore, we investigated the effect of PTEN on PI3K/Akt and VEGF/FLT1 pathways by transfecting wild-type PTEN gene to induce high expression of wild-type PTEN gene and protein in chronic myelogenous leukemia cell line K562 cells. We also observed the correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients. We found that the expression reconstitution of wild-type PTEN had significant effect on inhibiting proliferation, migration, and invasion abilities of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Down-regulation of PTEN expression accompanied by up-regulation of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. In conclusion, PTEN could modulate the function of VEGF/VEGFR signaling pathway down-regulation of Akt phosphorylation and that PTEN would be a candidate target to be addressed for inhibiting angiogenesis along with the treatment of myeloid leukemia.

Medullary thyroid carcinoma (MTC) occurs sporadically in 75% of patients. Metastatic disease is associated with significantly poorer survival. The aim of this study was to identify prognostic markers for progressive MTC and oncogenic factors associated with response to vandetanib therapy.

Clinical courses of 32 patients with sporadic MTC (n=10 pN0cM0, n=8 pN1cM0, n=14 pN1cM1) were compared with genetic profiles of the patients' primary tumour tissue. Analysis for RET proto-oncogene mutations was performed by Sanger sequencing and next-generation sequencing (NGS). The mRNA expression (mRNA count) of 33 targets was measured by nCounter NanoString analysis.

Somatic RET mutations occurred in 21/32 patients. The RET918 mutation was found in 8/14 pN1cM1 patients. BRAF (P=0.019), FGFR2 (P=0.007), FGFR3 (P=0.044) and VEGFC (P=0.042) mRNA expression was significantly lower in pN1cM0/pN1cM1 compared with pN0cM0 patients, whereas PDGFRA (P=0.026) mRNA expression was significantly higher in pN1cM0/pN1cM1 when compared with pN0cM0 patients. Among the 10/32 vandetanib-treated patients, 5 showed partial response (PR), all harbouring the RET918 mutation. mRNA expression of FLT1 (P=0.039), FLT4 (P=0.025) and VEGFB (P=0.042) was significantly higher in therapy responders.

In this study, we identified molecular markers in primary tumour tissue of sporadic MTC associated with the development of metastasis (both lymph node and organ metastasis) as well as response to vandetanib therapy.

A large number of pseudogenes have been found to be transcribed in human cancers. However, only a few pseudogenes are functionally characterized. Here, we identified a transcribed pseudogene of VEGFR1, or fms-related tyrosine kinase 1 (FLT1), in human colorectal cancer cells. Interestingly, this pseudogene (designated as FLT1P1) was found to be transcribed bidirectionally and functionally modulated cognate VEGFR1 protein expression in the cells. Mechanistically, expression of FLT1P1 antisense transcript not only inhibited the VEGFR1 expression, but also inhibited non-cognate VEGF-A expression through interaction with miR-520a. Perturbation of FLT1P1 expression by RNA interference (RNAi) markedly inhibited tumor cell proliferation and xenograft tumor growth. This study identifies FLT1P1 antisense as a critical regulator of VEGFR1 and VEGF-A expression in colorectal cancer cells, and highlights its role in regulation of the pathogenesis of colorectal cancer.

CONCLUSIONS

The VEGFR1 pseudogene, FLT1P1, is a novel and functional regulator of VEGF signaling and its targeting could be an alternative strategy to modulate its cognate/target gene expression and downstream activity in cancer.

Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and oedema, resolves on placental delivery. Its pathogenesis is thought to be associated to a hypoxic placenta. Placental hypoxia is responsible for the maternal vascular dysfunction via the increased placental release of anti-angiogenic factors such as soluble flt1 and endoglin. These soluble receptors bind VEGF, PLGF and TGFbeta1 and 3 in the maternal circulation, causing endothelial dysfunction in many maternal tissues. Despite these recent and important new molecular findings, it is important to consider that normal pregnancy is also characterized by systemic inflammation, oxidative stress and alterations in levels of angiogenic factors and vascular reactivity. Both the placenta and maternal vasculatures are major sources of reactive oxygen and nitrogen species which can produce powerful pro-oxidants that covalently modify proteins and alter vascular function in preeclampsia. Finally, the recent demonstration of activating auto-antibodies to the Angiotensin 1 receptor that experimentally play a major pathogenic role in preeclampsia further indicates the pleiotropism of aetiologies of this condition.

Background: Preeclampsia (PE) is a pregnancy-specific hypertension syndrome and is one of the leading causes of maternal and perinatal morbidity and mortality. Long noncoding RNAs (lncRNAs) have been reported to be abnormally expressed in many diseases, including preeclampsia. The present study is aimed at identifying the key genes and lncRNA-associated competing endogenous RNA (ceRNA) networks in early-onset preeclampsia (EOPE).

Methods: We investigated expression profiles of differentially expressed lncRNAs (DElncRNAs) and genes (DEGs) in placental tissues of EOPE and healthy controls with Human LncRNA Array v4. The potential functions of DEGs and DElncRNAs were predicted using the clusterProfiler package. The lncRNA-mRNA coexpression network was constructed via Pearson's correlation coefficient. The protein-protein interaction (PPI) network of DEGs was constructed, and the hub genes were obtained using the STRING database and Cytoscape. The ceRNA networks were constructed based on miRWalk and LncBase v2. qRT-PCR was performed to confirm the expression of lncRNA MIR193BHG, PROX1-AS1, and GATA3-AS1. ROC curves were performed to assess the clinical value of lncRNA MIR193BHG, PROX1-AS1, and GATA3-AS1 in the diagnosis of EOPE.

Results: We found 6 hub genes (SPP1, CCR2, KIT, ENG, ACKR1, and FLT1) altered in placental tissues of EOPE and constructed a ceRNA network, including 21 lncRNAs, 3 mRNAs, and 69 miRNAs. The expression of lncRNA MIR193BHG and GATA3-AS1 were elevated and showed good clinical values for diagnosing EOPE.

Conclusion: This study provides novel insights into the lncRNA-related ceRNA network in EOPE and identified two lncRNAs as potential prognostic biomarkers in EOPE.

Metabolic diseases, such as obesity and diabetes, are a serious burden for the health system. Vascular endothelial growth factor (VEGF)-B has been shown to regulate tissue uptake and accumulation of fatty acids and is thus involved in these metabolic diseases. However, the cell-type-specific expression pattern of Vegfb and its receptor (VEGFR1, gene Flt1) remains unclear. We explore the expression of Vegfb and Flt1 in the murine heart, lung and kidney by utilizing β-galactosidase knock-in mouse models and combining the analysis of reporter gene expression and immunofluorescence microscopy. Furthermore, Flt1 heterozygous mice were analyzed with regard to muscular fatty acid accumulation and peripheral insulin sensitivity. Throughout the heart, Vegfb expression was found in cardiomyocytes with a postnatal ventricular shift corresponding to known changes in energy requirements. Vegfb expression was also found in the pulmonary myocardium of the lung and in renal epithelial cells of the thick ascending limb of Henle's loop, the connecting tubule and the collecting duct. In all analyzed organs, VEGFR1 expression was restricted to endothelial cells. We also show that reduced expression of VEGFR1 resulted in decreased cardiac fatty acid accumulation and increased peripheral insulin sensitivity, possibly as a result of attenuated VEGF-B/VEGFR1 signaling. Our data therefore support a tightly controlled, paracrine signaling mechanism of VEGF-B to VEGFR1. The identified cell-specific expression pattern of Vegfb and Flt1 might form the basis for the development of cell-type-targeted research models and contributes to the understanding of the physiological and pathological role of VEGF-B/VEGFR1 signaling.

Microvascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an in vitro model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the γ-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a γ-secretase inhibitor or astrocyte conditioned medium.

Clear cell renal cell carcinoma (ccRCC) was the most aggressive histological type of renal cell carcinoma (RCC) and accounted for 70-80% of cases of all RCC. The aim of this study was to identify the potential biomarker in ccRCC and explore their underlying mechanisms. Four profile datasets were downloaded from the GEO database to identify DEGs. GO and KEGG analysis of DEGs were performed by DAVID. A protein-protein interaction (PPI) network was constructed to predict hub genes. The hub gene expression within ccRCC across multiple datasets and the overall survival analysis were investigated utilizing the Oncomine Platform and UALCAN dataset, separately. A meta-analysis was performed to explore the relationship between the hub genes: EGFR and ccRCC. 127 DEGs (55 upregulated genes and 72 downregulated genes) were identified from four profile datasets. Integrating the result from PPI network, Oncomine Platform, and survival analysis, EGFR, FLT1, and EDN1 were screened as key factors in the prognosis of ccRCC. GO and KEGG analysis revealed that 127 DEGs were mainly enriched in 21 terms and 4 pathways. The meta-analysis showed that there was a significant difference of EGFR expression between ccRCC tissues and normal tissues, and the expression of EGFR in patients with metastasis was higher. This study identified 3 importance genes (EGFR, FLT1, and EDN1) in ccRCC, and EGFR may be a potential prognostic biomarker and novel therapeutic target for ccRCC, especially patients with metastasis.

Cervical cancer and endometrial cancer remain serious threats to women's health. Even though some patients can be treated with surgery plus chemoradiotherapy as a conventional option, the overall efficacy is deemed unsatisfactory. As such, the development for new treatment approaches is truly necessary. In recent years, immunotherapy has been widely used in clinical practice and it is an area of great interest that researchers are keeping attention on. However, a thorough immune-related genes (IRGs) study for cervical cancer and endometrial cancer is still lacking. We therefore aim to make a comprehensive evaluation of IRGs through bioinformatics and large databases, and also investigate the relationship between the two types of cancer. We reviewed the transcriptome RNAs of IRGs and clinical data based on the TCGA database. Survival-associated IRGs in cervical/endometrial cancer were identified using univariable and multivariable Cox proportional-hazard regression analysis for developing an IRG signature model to evaluate the risk of patients. In the end, this model was validated based on the enrichment analyses through GO, KEGG, and GSEA pathways, Kaplan-Meier survival curve, ROC curves, and immune cell infiltration. Our results showed that out of 25/23 survival-associated IRGs for cervical/endometrial cancer, 13/12 warranted further examination by multivariate Cox proportional-hazard regression analysis and were selected to develop an IRGs signature model. As a result, enrichment analyses for high-risk groups indicated main enriched pathways were associated with tumor development and progression, and statistical differences were found between high-risk and low-risk groups as shown by Kaplan-Meier survival curve. This model could be used as an independent measure for risk assessment and was considered relevant to immune cell infiltration, but it had nothing to do with clinicopathological characteristics. In summary, based on comprehensive analysis, we obtained the IRGs signature model in cervical cancer (LTA, TFRC, TYK2, DLL4, CSK, JUND, NFATC4, SBDS, FLT1, IL17RD, IL3RA, SDC1, PLAU) and endometrial cancer (LTA, PSMC4, KAL1, TNF, SBDS, HDGF, LTB, HTR3E, NR2F1, NR3C1, PGR, CBLC), which can effectively evaluate the prognosis and risk of patients and provide justification in immunology for further researches.

Keywords: TCGA; cervical cancer; endometrial cancer; immune-related genes; prognostic model.

Efforts have been made to characterize the high-altitude adaption in Tibetan pigs and identified vast of genes or genomic regions undergone natural selection. Nonetheless, information concerning gene expression and DNA methylation changes response to low-altitude acclimation in Tibetan pigs is long overdue. To explore the exceptional mechanisms of gene expression and DNA methylation that are induced by low altitude environments in Tibetan pigs, we performed a comparative transcriptomic and DNA methylation analysis of skeletal muscle between indigenous Tibetan pigs that reside in high altitude regions (~4000m) and their counterparts that migrated to the geographically neighboring low-altitude regions (~500m) for nearly ten generations. Many genes that related to hypoxia response (EGLN3 and FLT1) and energy metabolism (TFB2M) were differentially expressed, but without significant DNA methylation changes. We also found genes embedded in differentially methylated regions were mainly involved in 'Starch and sucrose metabolism', 'glucuronosyltransferase activity' processes. Specifically, our results showed increased SIN3A mRNA expression, with hypomethylation status of its promoter, in longissimus dorsi muscle of low-altitude Tibetan pig. Another gene, CACNG6, showed decreasing expression level with an elevated methylation in its intron 3. These results indicated DNA-methylation-mediated expression alterations in low-altitude acclimation. We envision that this study will serve as a valuable resource for mammal acclimation research and agricultural food industry.

Endothelial progenitor cells (EPCs) participate in the neovascularization processes in the development of hepatocellular carcinoma (HCC). We investigated whether interactions between EPCs and HCC cells affect chemotactic and pro-inflammatory activities of EPCs. Two distinct phenotypes of circulating EPCs, i.e., myeloid-derived EPCs (colony forming unit-endothelial cells, CFU-ECs) and outgrowth EPCs (endothelial-colony forming cells, ECFCs), were co-cultured with Huh7 and Hep3B cells by using transwell chamber and IBIDI(TM) Culture-Inserts and μ-slide plates. Transwell and horizontal migration/invasion assays and time-lapse microscopy were used to monitor and analyze the migration and invasion of EPCs induced by these HCC cells. A human cytokine antibody array was used to compare protein expression profiles in EPCs and HCC cells. Flow cytometry and electromobility shift analysis were used to detect nuclear factor-κB (NF-κB)-DNA binding activity and pro-inflammatory adhesion molecule expression in EPCs. Ectopic full-length CC chemokine receptor 6 (CCR6) plasmid was used to transfect into ECFCs to investigate the role of CCR6 in HCC-induced EPC migration and invasion. The results show that co-culture with Huh7 and Hep3B cells induces the expression of endothelial cell (EC) markers KDR, Flt1, CD31 and VE-cadherin in CFU-ECs, but down-regulates the expressions of CD31 and VE-cadherin in ECFCs. These HCC cells induce migration and invasion of CFU-ECs, but not ECFCs, and do not affect the cell cycle distribution in these EPCs. Cytokine protein array identifies macrophage inflammatory protein-3α (MIP-3α) produced by HCC cells as a critical factor responsible for the HCC-induced chemotaxis of CFU-ECs, which highly express the specific MIP-3α counterreceptor CCR6. Overexpressing CCR6 in ECFCs significantly increases their chemotaxis in response to HCC cells. Co-culturing EPCs with HCC cells results in decreases in NF-κB binding activity and hence intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions in EPCs. Our results indicate that HCC cells exert differential effects on CFU-ECs and ECFCs, with increased chemotaxis for CFU-ECs, but not ECFCs. This HCC-induced chemotaxis of CFU-ECs is mediated by MIP-3α produced by HCC cells, which targets to CCR6 on CFU-ECs. Tumors may provide a humoral microenvironment to attenuate the pro-inflammatory activity of EPCs, which might be associated with the tumor escape mechanism.

BACKGROUND

Pre-eclampsia (PE) is a hypertensive multisystem disorder, causing significant fetal-maternal mortality and morbidity worldwide. This study aims to define possible longitudinal predictive mRNA markers involved in the main pathogenic pathways of PE: inflammation [macrophage migration inhibitory factor (MIF)], hypoxia and oxidative stress [hypoxia inducible factor 1-α subunit (HIF1A) and β-site APP-cleaving enzyme-2 (BACE2)] and endothelial dysfunction [endoglin (ENG), fms-related tyrosine kinase-1 (FLT1) and vascular endothelial growth factor (VEGF)].

METHODS

Peripheral blood was collected from 33 singleton pregnancies characterized by a high cardiovascular profile risk sampled consecutively at 6-16; 17-23; 24-30; 31-34; ≥35 weeks followed by the Obstetrics and Gynecology Unit of the San Raffaele Hospital in Milan. A real-time quantitative PCR reaction was performed on plasma RNA.

RESULTS

Of the 33 women enrolled, nine developed PE. Until 23 weeks HIF1A was significantly higher in women who later developed PE compared to women who did not (p=0.049 and p=0.012 in the first and second blood collection). In the third time interval MIF (p=0.0005), FLT1 (p=0.024), ENG (p=0.0034) and BACE2 (p=0.044) appeared to be significantly increased while HIF1A was elevated even from 24 week onwards but not reaching the statistical significance. In the fourth time interval ENG mRNA still remained increased (p=0.037).

CONCLUSIONS

HIF1A, marker of hypoxia and oxidative stress, and MIF, marker of inflammation, seemed to be the most promising RNA markers, suggesting that hypoxia, principally, and inflammation may play an important role in PE pathogenesis.

The therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) have recently increased due to the development of tyrosine kinase inhibitors (TKIs), some of which have achieved remarkable clinical responses in MTC patients. However, the molecular basis for the large variability in TKI responses is unknown. In this exploratory study, we investigated the expression of eight key TKI target proteins (EGFR, KIT, MET, PDGFRB, VEGF (VEGFA), VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4)) by immunohistochemistry in 103 molecularly characterized MTC samples and identified the associated clinical and molecular features. A number of MTC samples exhibited a high expression of VEGFR2 and VEGFR3, which were overexpressed in 57 and 43% of the MTC samples respectively. VEGFR1, PDGFRB, VEGF, KIT, and MET were present in 34-20% of the cases, while EGFR was highly expressed in only 10% of the MTC samples. Some proteins exhibited large differences in expression between sporadic and familial cases, suggesting that different RET mutations may be associated with the immunohistochemical profiles. MTC samples with the C634 RET mutation exhibited a higher expression of VEGFR3 and KIT than the M918T RET-mutated and non-mutated RET tumor samples (P=0.005 and P=0.007 respectively) and a lower expression of VEGFR1 (P=0.04). Non-mutated RET MTC cases exhibited a lower expression of PDGFRB (P=0.04). Overall, this is the first study, to our knowledge, to show that multiple TKI targets are highly expressed in a subset of MTCs, suggesting that molecular stratification of patients may have the potential to improve TKI therapies for MTC.

Anti-VEGF antibody bevacizumab has prolonged progression-free survival in several cancer types, however acquired resistance is common. Adaption has been observed pre-clinically, but no human study has shown timing and genes involved, enabling formulation of new clinical paradigms. In a window-of-opportunity study in 35 ductal breast cancer patients for 2weeks prior to neoadjuvant chemotherapy, we monitored bevacizumab response by Dynamic Contrast-Enhanced Magnetic Resonance [DCE-MRI], transcriptomic and pathology. Initial treatment response showed significant overall decrease in DCE-MRI median K(trans), angiogenic factors such ESM1 and FLT1, and proliferation. However, it also revealed great heterogeneity, spanning from downregulation of blood vessel density and central necrosis to continued growth with new vasculature. Crucially, significantly upregulated pathways leading to resistance included glycolysis and pH adaptation, PI3K-Akt and immune checkpoint signaling, for which inhibitors exist, making a strong case to investigate such combinations. These findings support that anti-angiogenesis trials should incorporate initial enrichment of patients with high K(trans), and a range of targeted therapeutic options to meet potential early resistance pathways. Multi-arm adaptive trials are ongoing using molecular markers for targeted agents, but our results suggest this needs to be further modified by much earlier adaptation when using drugs affecting the tumor microenvironment.

Flt1 and Flk1 are receptor tyrosine kinases for vascular endothelial growth factor-A which play a crucial role in physiological and pathological angiogenesis. To study genetic interaction between the Flt1 and Flk1 genes, we crossed between Flt1 and Flk1 heterozygous (Flt1(+/-) and Flk1(+/-)) mice. We found that Flt1; Flk1 double heterozygous (Flt1(+/-); Flk1(+/-)) mice showed enlarged eyes similar to the buphthalmia detected in human congenital glaucoma with elevation of intraocular pressure (IOP). Actually, IOP was elevated in Flt1(+/-); Flk1(+/-) mice and also in Flt1 or Flk1 single heterozygous mice. However, none of these mutants showed hallmarks of glaucoma such as ganglion cell death and excavation of optic disc. To clarify the pathological causes for enlarged eyes and elevated IOP, we investigate the mice from matings between Flt1(+/-) and Flk1(+/-) mice. Flt1(+/-) mice showed enlarged Schlemm's canal and disordered collagen fibers in the sclera, whereas Flk1(+/-) mice showed atrophied choriocapillaris in the choroid. These tissues are a part of the main outflow and alternative uveoscleral outflow pathway of the aqueous humor, suggesting that these pathological changes found in Flt1(+/-) and Flk1(+/-) mice are associated with the buphthalmia in Flt1(+/-); Flk1(+/-) mice.

Preeclampsia risk is influenced by both the mother's genetic background and the genetics of her fetus; however, the specific genes responsible for conferring preeclampsia risk have largely remained elusive. Evidence that preeclampsia has a genetic predisposition was first detailed in the early 1960s, and overall preeclampsia heritability is estimated at ∼55%. Many traditional gene discovery approaches have been used to investigate the specific genes that contribute to preeclampsia risk, but these have largely not been successful or reproducible. Over the past decade, genome-wide association studies have allowed for significant advances in the understanding of the genetic basis of many common diseases. Genome-wide association studies are predicated on the idea that the genetic basis of many common diseases are complex and polygenic with many variants, each with modest effects that contribute to disease risk. Using this approach in preeclampsia, a large genome-wide association study recently identified and replicated the first robust fetal genomic region associated with excess risk. A screen of >7 million genetic variants in 2658 offspring from preeclamptic women and 308,292 population controls identified a single association signal close to the Fms-like tyrosine kinase 1 gene, on chromosome 13. Fms-like tyrosine kinase 1 encodes soluble Fms-like tyrosine kinase 1, a splice variant of the vascular endothelial growth factor receptor that exerts antiangiogenic activity by inhibiting signaling of proangiogenic factors. The Fms-like tyrosine kinase 1 pathway is central in preeclampsia pathogenesis because excess circulating soluble Fms-like tyrosine kinase 1 in the maternal plasma leads to the hallmark clinical features of preeclampsia, including hypertension and proteinuria. The success of this landmark fetal preeclampsia genome-wide association study suggests that well-powered, larger maternal and fetal genome-wide association study will be fruitful in identifying additional common variants that implicate causal preeclampsia genes and pathways. Such efforts will rely on the continued development of large preeclampsia consortia focused on preeclampsia genetics to obtain adequate sample sizes, detailed clinical phenotyping, and matched maternal-fetal samples. In summary, the fetal preeclampsia genome-wide association study represents an exciting advance in preeclampsia biology, suggesting that dysregulation at the Fms-like tyrosine kinase 1 locus in the fetal genome (likely in the placenta) is a fundamental molecular defect in preeclampsia.

Preeclampsia is an important pregnancy disorder with serious maternal and fetal complications which its etiology has not been completely understood yet. Early diagnosis and management of disease could reduce its potential side effects. The vascular endothelial growth factor (VEGF) family including VEGF-A is the most potent endothelial growth factor which induces angiogenesis and endothelial cell proliferation and has basic role in vasculogenesis. VEGF and its tyrosine kinase receptors (Flt1 and KDR) are major factors for fetal and placental angiogenic development. Finding mechanisms involved in expression of angiogenic factors may lead to new prognostic and therapeutic points in management of preeclampsia. Recent researches, has shown capability of some anti-angiogenic factors as potential candidate to be used as early predictors for preeclampsia. Soluble fms-like tyrosin kinase-1 (sFlt1) is a truncated splice variant of the membrane-bound VEGF receptor Flt1, that is produced by the placenta and it can bind to angiogenic growth factors and neutraliz, their effects. It is also observed that the ratio of sFlt1 to placental growth factor is valuable as prognostic marker. In this review, VEGF family member's role in angiogenesis is evaluated as biomarkers to be used for prediction of preeclampsia.

BACKGROUND

Preservation induced injury is a major contributing factor to early graft dysfunction in liver allograft recipients. We hypothesized that changes in gene expression represent the earliest indicator of ischemia/reperfusion-related injuries measurable in the graft and could be used as prognostic marker for the occurrence of graft-related complications.

METHODS

We studied the expression of 67 genes, known to play a role in acute inflammatory processes by real-time polymerase chain reaction in 59 postperfusion biopsies. The level of expression was correlated with the occurrence of graft-related complications.

RESULTS

We identified six genes that were significantly correlated with the occurrence of early graft dysfunction (Spearman test, two-tailed; P<0.05). High C-reactive protein (CRP) gene expression levels correlated significantly with the need of therapeutic interventions due to graft-related complications (P=0,011). Furthermore, five genes related to vascular endothelial cell physiology (CTGF, WWP2, CD274, VEGF. and its receptor FLT1) showed significantly reduced expression in the postperfusion biopsies of patients with need of therapeutic interventions due to graft-related complications in the first month (P<0.05). Using a risk score based on the expression of these five genes, complications could be predicted with 96% sensitivity (ROC analysis, specificity: 74%, positive predictive value: 72%, negative predictive value: 96%).

CONCLUSIONS

Quantitative gene expression analysis in postperfusion biopsies may be a valuable tool to prospectively identify patients at risk for early clinical allograft dysfunction after liver transplantation.

PurposePlacental growth factor (PlGF) is a member of the VEGF family that plays an important role in experimental models of diabetic retinopathy and retinal neovascularization. We aimed to investigate whether vitreous levels of PlGF correlated with proliferative diabetic retinopathy (PDR) status, VEGF levels, and bevacizumab treatment. We also analysed PDR membranes to confirm the presence of the PlGF receptor, FLT1, in endothelial cells.MethodsThis was a case-control study: undiluted vitreous fluid samples were obtained from 28 active PDR patients without preoperative bevacizumab treatment, 21 active PDR patients with preoperative bevacizumab treatment, 18 inactive PDR patients, and 21 control patients. PlGF and VEGF levels in samples were determined by enzyme-linked immunosorbent assay. Immunohistochemistry for FLT1 was performed on human PDR membranes.ResultsCompared to control, vitreous PlGF levels were higher in both active PDR without bevacizumab (P<0.0001) and with bevacizumab (P<0.0001). There was no significant difference in PlGF between active PDR patients without and with bevacizumab (P=0.56). Compared to active PDR, PlGF levels were significantly reduced in inactive PDR (P=0.004). PlGF levels were highly correlated with VEGF levels in active PDR. VEGFR1 was expressed in endothelial cells in human PDR membranes.ConclusionThe strong correlation of PlGF levels with PDR disease status and expression of FLT1 in human PDR membranes suggest that PlGF has a pathogenic role in proliferative diabetic retinopathy. Therapeutic targeting of PlGF with agents like aflibercept may be beneficial.

BACKGROUND

Podocytes are major components of the filtration barrier and a renal source of vascular endothelial growth factor (VEGF). Chronic renovascular disease (RVD) progressively degrades the renal function, accompanied by podocyte damage and a progressive reduction in VEGF. We showed that the endothelin (ET) pathway contributes to this pathological process and ET-A (but not ET-B) receptor antagonism protects the kidney in RVD. We hypothesize that ET-A-induced renoprotection is largely driven by the protection of podocyte integrity and function.

METHODS

To mimic the renal environment of chronic RVD, human podocytes were incubated under chronic hypoxia for 96 h and divided in untreated or treated with an ET-A or ET-B receptor antagonist. Cells were quantified after 96 h. Cell homogenates and media were obtained after 1, 24 and 96 h to quantify production of VEGF, anti-VEGF soluble receptor s-Flt1, and the expression of apoptotic mediators. A separate set of similar experiments was performed after addition of a VEGF-neutralizing antibody (VEGF-NA).

RESULTS

Hypoxia decreased podocyte number, which was exacerbated by ET-B but improved after ET-A antagonism. Production of VEGF was preserved by ET-A antagonism, whereas s-Flt1 increased in hypoxic cells after ET-B antagonism only, accompanied by a greater expression of pro-apoptotic mediators. On the other hand, treatment with VEGF-NA diminished ET-A-induced protection of podocytes.

CONCLUSIONS

ET-A antagonism preserves podocyte viability and integrity under chronic hypoxia, whereas ET-B antagonism exacerbates podocyte dysfunction and death. Enhanced bioavailability of VEGF after ET-A antagonism could be a pivotal mechanism of podocyte protection that significantly contributes to ET-A receptor blockade-induced renal recovery in chronic RVD.

Genetic Analysis Workshop 17 used real sequence data from the 1000 Genomes Project and simulated phenotypes influenced by a large number of rare variants. Our aim is to evaluate the performance of various collapsing methods that were developed for analysis of multiple rare variants. We apply collapsing methods to continuous phenotypes Q1 and Q2 for all 200 replicates of the unrelated individuals data. Within each gene, we collapse (1) all SNPs, (2) all SNPs with minor allele frequency (MAF) < 0.05, and (3) nonsynonymous SNPs with MAF < 0.05. We consider two tests when collapsing variants: using the proportion of variants and using the presence/absence of any variant. We also compare our results to a single-marker analysis using PLINK. For phenotype Q1, the proportion test for collapsing rare nonsynonymous SNPs often performed the best. Two genes (FLT1 and KDR) had statistically significant results. A single-marker analysis using PLINK also provided statistically significant results for some SNPs within these two genes. For phenotype Q2, collapsing rare nonsynonymous SNPs performed the best, with almost no difference between proportion and presence tests. However, neither collapsing methods nor a single-marker analysis provided statistically significant results at the true genes for Q2. We also found that a large number of noncausal genes had high correlations with causal genes for Q1 and Q2, which may account for inflated false positives.