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Abstract Endometriosis, a prevalent condition, has long been recognized as a chronic and debilitating ailment affecting an estimated 1790 million women worldwide. Observational studies have established a correlation between endometriosis and ovarian cancer. Thus, we endeavored to employ Two-Sample Mendelian Randomization, utilizing summary statistics from a Genome-Wide Association Study of endometriosis and epithelial ovarian cancer, with genetic markers serving as proxies for epithelial ovarian cancer. The analysis revealed a significant correlation between these entities, with an odds ratio (OR) of 1.23 (95% CI 1.11–1.36). Upon histotype-specific examination, robust evidence emerged for an association of endometriosis with the risk of endometrioid carcinoma (OR 1.49, 95% CI 1.24–1.81), clear cell carcinoma (OR 2.56, 95% CI 1.75–3.73), and low malignant potential tumors (OR 1.28, 95% CI 1.08–1.53). These findings provide a theoretical framework for prospective investigations aimed at enhancing the potential therapeutic efficacy of managing endometriosis in averting the onset and progression of ovarian cancer.

Abstract Age is closely related to human health and disease risks. However, chronologically defined age often disagrees with biological age, primarily due to genetic and environmental variables. Identifying effective indicators for biological age in clinical practice and self-monitoring is important but currently lacking. The human lens accumulates age-related changes that are amenable to rapid and objective assessment. Here, using lens photographs from 20 to 96-year-olds, we develop LensAge to reflect lens aging via deep learning. LensAge is closely correlated with chronological age of relatively healthy individuals (R2 > 0.80, mean absolute errors of 4.25 to 4.82 years). Among the general population, we calculate the LensAge index by contrasting LensAge and chronological age to reflect the aging rate relative to peers. The LensAge index effectively reveals the risks of age-related eye and systemic disease occurrence, as well as all-cause mortality. It outperforms chronological age in reflecting age-related disease risks (p

Abstract Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

Platelet-derived growth factor-D (PDGF-D) is abundantly expressed in ocular diseases. Yet, it remains unknown whether and how PDGF-D affects ocular cells or cell-cell interactions in the eye. In this study, using single-cell RNA sequencing (scRNA-seq) and a mouse model of PDGF-D overexpression in retinal pigment epithelial (RPE) cells, we found that PDGF-D overexpression markedly upregulated the key immunoproteasome genes, leading to increased antigen processing/presentation capacity of RPE cells. Also, more than 6.5-fold ligand-receptor pairs were found in the PDGF-D overexpressing RPE-choroid tissues, suggesting markedly increased cell-cell interactions. Moreover, in the PDGF-D-overexpressing tissues, a unique cell population with a transcriptomic profile of both stromal cells and antigen-presenting RPE cells was detected, suggesting PDGF-D-induced epithelial-mesenchymal transition of RPE cells. Importantly, administration of ONX-0914, an immunoproteasome inhibitor, suppressed choroidal neovascularization (CNV) in a mouse CNV model in vivo. Together, we show that overexpression of PDGF-D increased pro-angiogenic immunoproteasome activities, and inhibiting immunoproteasome pathway may have therapeutic value for the treatment of neovascular diseases.

Tumor blood vessels contribute to cancer growth, invasion and metastasis. Here, by using single cell transcriptomics, the authors report an inventory of endothelial cell heterogeneity in patients with breast cancer, including a subtype that expresses genes involved in lipid processing and is regulated by PPAR-γ.

Abstract Angiogenesis is the process of new blood vessel growth from pre‐existing ones involving vascular endothelial cell activation, proliferation, migration, and tube formation. The vascular endothelial growth factor A (VEGF‐A) is known to be a key factor that promotes angiogenesis. Pathological angiogenesis is a key and common feature of numerous ocular neovascular diseases, such as wet age‐related macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, and corneal neovascularization. Although anti‐VEGF treatment has been a great success for the treatment of such ocular neovascular diseases, many challenges exist, such as limited efficacy, unresponsiveness in many patients, drug resistance, treatment burden due to repeated intravitreous injection, all of which incent much effort and enthusiasm to find new and better treatment for ocular neovascular diseases. In recent years, new antiangiogenic drug, targets and drug delivery methods have been developed. This perspective discusses the status of currently available therapies, challenges, opportunities, and potential new directions toward better therapies for ocular neovascular diseases.

This review discusses our current understanding of chromatin biology and bioinformatics under the unifying concept of “chromatin hubs.” The first part reviews the biology of chromatin hubs, including chromatin–chromatin interaction hubs, chromatin hubs at the nuclear periphery, hubs around macromolecules such as RNA polymerase or lncRNAs, and hubs around nuclear bodies such as the nucleolus or nuclear speckles. The second part reviews existing computational methods, including enhancer–promoter interaction prediction, network analysis, chromatin domain callers, transcription factory predictors, and multi-way interaction analysis. We introduce an integrated model that makes sense of the existing evidence. Understanding chromatin hubs may allow us (i) to explain long-unsolved biological questions such as interaction specificity and redundancy of mechanisms, (ii) to develop more realistic kinetic and functional predictions, and (iii) to explain the etiology of genomic disease.

Computational prediction of cell-cell interactions (CCIs) is becoming increasingly important for understanding disease development and progression. We present a benchmark study of available CCI prediction tools based on single-cell RNA sequencing (scRNA-seq) data. By comparing prediction outputs with a manually curated gold standard for idiopathic pulmonary fibrosis (IPF), we evaluated prediction performance and processing time of several CCI prediction tools, including CCInx, CellChat, CellPhoneDB, iTALK, NATMI, scMLnet, SingleCellSignalR, and an ensemble of tools. According to our results, CellPhoneDB and NATMI are the best performer CCI prediction tools, among the ones analyzed, when we define a CCI as a source-target-ligand-receptor tetrad. In addition, we recommend specific tools according to different types of research projects and discuss the possible future paths in the field.

Amblyopia is a common eye disease characterized by impaired best-corrected visual acuity. It starts in early childhood and leads to permanent vision reduction if left untreated. Even though many young patients with amblyopia are well treated in clinical practice, the underlying mechanism remains to be elucidated, which limits not only our understanding of this disease but also the therapeutic approach. To investigate the molecular mechanism of amblyopia, primate and rodent models of monocular-deprived amblyopia were created for mRNA screening and confirmation. We obtained 818 differentially expressed genes from the dorsal lateral geniculate nucleus (dLGN) of a primate model of amblyopia. After Gene Ontology and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses, the main enriched pathways were related to neural development. Interestingly, a particular neurotransmitter pathway, the dopaminergic pathway, was identified. The downregulation of dopamine receptor D1 (DRD1) was confirmed in both monkey and mouse samples. Furthermore, the immunofluorescence staining indicated that DRD1 expression was downregulated in both ventrolateral region of the contralateral dLGN and the dorsomedial region of the ipsilateral dLGN in the mouse model. The regions with downregulated expression of DRD1 were the downstream targets of the visual projection from the amblyopic eye. This study suggested that the downregulation of DRD1 in the LGN may be a cause for amblyopia. This may also be a reason for the failure of some clinical cases of levodopa combined with carbidopa applied to amblyopes.

Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for scientists of many research disciplines due to its ability to elucidate the heterogeneous and complex cell-type compositions of different tissues and cell populations. Traditional cell-type identification methods for scRNA-seq data analysis are time-consuming and knowledge-dependent for manual annotation. By contrast, automatic cell-type identification methods may have the advantages of being fast, accurate, and more user friendly. Here, we discuss and evaluate thirty-two published automatic methods for scRNA-seq data analysis in terms of their prediction accuracy, F1-score, unlabeling rate and running time. We highlight the advantages and disadvantages of these methods and provide recommendations of method choice depending on the available information. The challenges and future applications of these automatic methods are further discussed. In addition, we provide a free scRNA-seq data analysis package encompassing the discussed automatic methods to help the easy usage of them in real-world applications.

Introduction Tobacco use poses a threat to the health of pregnant women and their children. Our study assessed the association between maternal exposure to secondhand smoke (SHS) during pregnancy and children’s cerebral palsy (CP) in Shandong, China. Methods In our observational study, 5067 mother–child pairs were included from Shandong Province, China. Mothers filled in questionnaires about exposure to SHS during pregnancy. Statistical analysis and logistic regression models were built in R program to estimate the association in adjusted odds ratio (AOR) between SHS exposure during pregnancy and risk of children’s CP, after adjustment for potential confounders including delivery mode and baby’s birthweight. Results Exposure to SHS was noted among 3663 (72.3%) of the 5067 non-smoking mothers during their pregnancy. Of the 239 CP children within the study, 192 (80.3%) were exposed to SHS during pregnancy. Children born to mothers exposed to SHS during pregnancy had a higher risk of CP (AOR=1.44; 95% CI: 1.02–2.04) than those born to non-exposed mothers, the risk increased by exposure time in the logistic regression model. The association between SHS exposure during pregnancy and CP children remained significant when adjusting for delivery mode and infant’s birthweight due to their significant association with CP, with an AOR of 1.46 (95% CI: 1.13–1.91) for 1–4 days/week and 1.63 (95% CI: 1.22–2.01) for 5–7 days/week exposure to SHS. Conclusions Our study suggests that maternal exposure to secondhand smoke during pregnancy is associated with children’s CP. Future preventive interventions of CP should include strategies that target the antenatal women who are exposed to SHS.

Anti-VEGF therapy has many limitations that might be resolved by using combination treatment approaches. Here, the authors demonstrate that the dual-targeting of VEGF and PDGF is required for targeting resistant FGF2+ tumors which depend on the recruitment of pericytes on tumor microvessels.

Platelet-derived growth factor C (PDGF-C) is a relatively new member of the PDGF family, discovered nearly 20 years after the finding of platelet-derived growth factor A (PDGF-A) and platelet-derived growth factor B (PDGF-B). PDGF-C is generally expressed in most organs and cell types. Studies from the past 20 years have demonstrated critical roles of PDGF-C in numerous biological, physiological and pathological processes, such as development, angiogenesis, tumour growth, tissue remodelling, wound healing, atherosclerosis, fibrosis, stem/progenitor cell regulation and metabolism. Understanding PDGF-C expression and activities thus will be of great importance to various research disciplines. In this review, however, we mainly discuss the expression and functions of PDGF-C and its receptors in development and stem cells.

Summary: Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting.For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).

Summary: Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting.For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Although JAM-C is abundantly expressed in the retinae and upregulated in choroidal neovascularization (CNV), it remains thus far poorly understood whether it plays a role in the blood-retinal barrier, which is critical to maintain the normal functions of the eye. Here, we report that JAM-C is highly expressed in retinal capillary endothelial cells (RCECs), and VEGF or PDGF-C treatment induced JAM-C translocation from the cytoplasm to the cytomembrane. Moreover, JAM-C knockdown in RCECs inhibited the adhesion and transmigration of macrophages from wet age-related macular degeneration (wAMD) patients to and through RCECs, whereas JAM-C overexpression in RCECs increased the adhesion and transmigration of macrophages from both wAMD patients and healthy controls. Importantly, the JAM-C overexpression-induced transmigration of macrophages from wAMD patients was abolished by the administration of the protein kinase C (PKC) inhibitor GF109203X. Of note, we found that the serum levels of soluble JAM-C were more than twofold higher in wAMD patients than in healthy controls. Mechanistically, we show that JAM-C overexpression or knockdown in RCECs decreased or increased cytosolic Ca2+ concentrations, respectively. Our findings suggest that the dynamic translocation of JAM-C induced by vasoactive molecules might be one of the mechanisms underlying inner endothelial BRB malfunction, and inhibition of JAM-C or PKC in RCECs may help maintain the normal function of the inner BRB. In addition, increased serum soluble JAM-C levels might serve as a molecular marker for wAMD, and modulating JAM-C activity may have potential therapeutic value for the treatment of BRB malfunction-related ocular diseases.

Retinal pigment epithelium (RPE), a postmitotic monolayer located between the neuroretina and choroid, supports the retina and is closely associated with vision loss diseases such as age-related macular degeneration (AMD) upon dysfunction. Although environmental stresses are known to play critical roles in AMD pathogenesis and the roles of other stresses have been well investigated, glucose deprivation, which can arise from choriocapillary flow voids, has yet to be fully explored. In this study, we examined the involvement of VEGFR2 in glucose deprivation-mediated cell death and the underlying mechanisms. We found that VEGFR2 levels are a determinant for RPE cell death, a critical factor for dry AMD, under glucose deprivation. RNA sequencing analysis showed that upon VEGFR2 knockdown under glucose starvation, endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are reduced. Consistently, VEGFR2 overexpression increased ER stress under the same condition. Although VEGFR2 was less expressed compared to EGFR1 and c-Met in RPE cells, it could elicit a higher level of ER stress induced by glucose starvation. Finally, downregulated VEGFR2 attenuated the oxidative stress and inflammatory factor expression, two downstream targets of ER stress. Our study, for the first time, has demonstrated a novel role of VEGFR2 in RPE cells under glucose deprivation, thus providing valuable insights into the mechanisms of AMD pathogenesis and suggesting that VEGFR2 might be a potential therapeutic target for AMD prevention, which may impede its progression.

Summary: Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues.For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).

Platelet-derived growth factor-D (PDGF-D) is highly expressed in immune cells. However, the potential role of PDGF-D in immune system remains thus far unclear. Here, we reveal a novel function of PDGF-D in activating both classical and alternative complement pathways that markedly increase chemokine and cytokine responses to promote macrophage polarization. Pharmacological targeting of the complement C3a receptor using SB290157 alleviated PDGF-D-induced neuroinflammation by blocking macrophage polarization and inhibited pathological choroidal neovascularization. Our study thus suggests that therapeutic strategies targeting both PDGF-D and the complement system may open up new possibilities for the treatment of neovascular diseases.

Abstract Background Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice. Methods In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry. Results In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC. Conclusion In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF/VEGF (vascular endothelial growth factor) family, which includes proteins that are well known for their mitogenic effects on multiple cell types. Glycosylation is one of the most important forms of posttranslational modification that has a significant impact on secreted and membrane proteins. Glycosylation has many well-characterized roles in facilitating protein processing and contributes to appropriate folding, conformation, distribution, and stability of proteins that are synthesized intracellularly in the endoplasmic reticulum (ER) and Golgi apparatus. Although the general process and functions of glycosylation are well documented, there are most likely others yet to be discovered, as the glycosylation of many potential substrates has not been characterized. In this study, we report that the PDGF-C protein is glycosylated at three sites, including Asn25, Asn55, and Asn254. However, we found that mutations at any of these sites do not affect the protein expression or secretion. Similarly, disruption of PDGF-C glycosylation had no impact on its progression through the ER and Golgi apparatus. However, the introduction of a mutation at Asn254 (N254 A) prevents the activation of full-length PDGF-C and its capacity for signaling via the PDGF receptor. Our findings reveal that glycosylation affects PDGF-C activation rather than the protein synthesis or processing. This study characterizes a crucial modification of the PDGF-C protein, and may shed new light on the process and function of glycosylation.

The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.

White adipocytes can be stimulated to express thermogenic genes in a process known as beiging. Here, the authors show that miR-327 is downregulated during beiging, which releases FGF10 from inhibition and supports beige adipocyte formation via signaling through FGFR2.

Cold-induced activation of thermogenesis in white adipose tissue (WAT), or ‘beiging’, is associated with WAT angiogenesis. Here the authors show that PDGF-CC is secreted from endothelial cells in the context of WAT angiogenesis and its paracrine action on adipocytes contributes to cold-induced beiging.

Introduction Our aim was to investigate the association between third-hand smoke exposure (THS) during pregnancy and postpartum depression (PPD) among Chinese women. Methods A total of 973 participants that completed the questionnaire and reviews were included in this cross-sectional study. The THS exposure was assessed based on a questionnaire with key questions from the Global Adult Tobacco Survey (2nd edition) while postpartum depression status was assessed using the Edinburgh Postnatal Depression Scale. Logistic regression models were used to estimate the association between THS exposure during pregnancy and risk of PPD, after adjustment for potential confounders. Results The prevalence of postpartum depression among all participants was 17.8%. Of the 973 participants, 725 (74.5%) were exposed to THS during pregnancy while 248 (25.5%) were not. Compared with those who were never exposed to passive smoking, puerperal women who were exposed to THS were at higher risk of PPD (OR=1.71, 95% CI: 1.12–2.60) in the logistic regression model. Conclusions Our study suggests that exposure to third-hand smoke may be a risk factor for postpartum depression among Chinese women. Future preventive interventions should include strategies that target the puerperal women who are exposed to THS during pregnancy. Tobacco control measures that are effective in reducing the prevalence of smoking may have little effect in reducing women’s exposure to THS during pregnancy, especially in private places that urgently need other public health strategies.

BACKGROUND:The severity of ischemic stroke symptoms varies among patients and is a critical determinant of patient outcome. To date, the association between the number of stenotic intracranial arteries and stroke severity remains unclear. AIMS:We aimed to investigate the association between the number of stenotic major intracranial arteries (NSMIA) and ischemic stroke severity, as well as the degree of stenosis and common stroke risk factors. METHODS:We performed a retrospective analysis of patients with digital subtraction angiography (DSA)-confirmed ischemic stroke. Clinical stroke severity was measured using the National Institutes of Health Stroke Scale (NIHSS). The number of stenotic vessels was counted from the internal carotid arteries and vertebral arteries, bilaterally. RESULTS:Eighty three patients were recruited from a single center and included in the study. NSMIA was significantly correlated with stroke severity (Pearson Correlation Coefficient = 0.485, P < 0.001), but not with the degree of stenosis (Pearson Correlation Coefficient = 0.01, P = 0.90). Multivariate regression analysis revealed that NSMIA was significantly associated with the NIHSS score after adjusting for stroke risk factors. The adjusted odds ratio (per lateral) was 2.092 (95% CI, 0.865 to 3.308, P = 0.001). The degree of stenosis was also significantly associated with the NIHSS score after adjusting for common risk factors. The odds ratio (per 10%) was 0.712 (95% CI, 0.202 to 1.223, P = 0.007). CONCLUSIONS:The number of stenotic intracranial major arteries is associated with the severity of ischemic stroke independent of the degree of stenosis and other stroke risk factors. To the best of our knowledge, this has not been previosuly studied in great detail using DSA. Our data highlight the importance of examining all major arteries in stroke patients.

The p53 upregulated modulator of apoptosis (PUMA) is known as an essential apoptosis inducer. Here, we report the seemingly paradoxical finding that PUMA is a proangiogenic factor critically required for the proliferation and survival of vascular and microglia cells. Strikingly, Puma deficiency by genetic deletion or small hairpin RNA knockdown inhibited developmental and pathological angiogenesis and reduced microglia numbers in vivo, whereas Puma gene delivery increased angiogenesis and cell survival. Mechanistically, we revealed that PUMA plays a critical role in regulating autophagy by modulating Erk activation and intracellular calcium level. Our findings revealed an unexpected function of PUMA in promoting angiogenesis and warrant more careful investigations into the therapeutic potential of PUMA in treating cancer and degenerative diseases.