Prospero Homeobox Research Articles

Genome resequencing reveals genetic status of Macrobrachium rosenbergii populations in China

The giant freshwater prawn (Macrobrachium rosenbergii) has been widely cultured in China since 1990s. In this study, 189 individuals were sampled from 10 distinct populations, including original populations introduced from abroad and domesticated strains in China, namely Florida (FD), "Nantaihu No.2" (NT), Vietnam (YN), Sri Lanka (SK), Bangladesh (ML), Thailand (TG), Taiwan (TW), "Shufeng No.1" (SF), Zhengda (ZD) and New Myanmar (NMD). The genetic diversity and population structure of these populations were analyzed based on a dataset of 300,828 genome-wide SNP loci. The population structure analysis showed that the 10 populations shared 4 ancestors, and the domestic populations (i.e., SF, ZD, TW and NT) shared a common ancestor. The phylogenetic analysis showed that the 10 populations were grouped into 4 branches, with all the domestic populations clustering together. Estimation of fixation index (Fst) values revealed relatively lower Fst values among the domestic populations, in contrast to the more significant differences observed between domestic and foreign-origin populations. Gene flow analysis revealed three significant events of genetic exchange among the 10 populations: (1) from YN to the common ancestor of FD, TG and the domestic populations, (2) from NMD to the common ancestor of TG and FD, and (3) from the common ancestor of NMD, YN, ML and SK to ZD population. In addition, selective sweep analysis, utilizing Fst and nucleotide diversity (Pi) estimations, was conducted on representative populations (i.e., ML and SF), and 7 positively selected genes were identified, including Prospero Homeobox 1 (Prox1), Forkhead Box L1 (Foxl1), Tousled Like Kinase 2 (TLK2), G Protein Subunit Alpha 14 (Gna14), Uronyl 2-sulfotransferase (Ust), MAX Network Transcriptional Repressor (Mnt) and Importin-9 (Ipo9). These findings contribute to elucidating the breeding potential of the introduced populations and provide insights into the genetic status of the domestic breeding strains of M. rosenbergii in China.

Prox1a promotes liver growth and differentiation by repressing cdx1b expression and intestinal fate transition in zebrafish

The liver is a key endoderm-derived multifunctional organ within the digestive system. Prospero homeobox 1 (Prox1) is an essential transcription factor for liver development, but its specific function is not well understood. Here, we show that hepatic development, including the formation of intrahepatic biliary and vascular networks, is severely disrupted in prox1a mutant zebrafish. We find that Prox1a is essential for liver growth and proper differentiation but not required for early hepatic cell fate specification. Intriguingly, prox1a depletion leads to ectopic initiation of a Cdx1b-mediated intestinal program and the formation of intestinal lumen-like structures within the liver. Morpholino knockdown of cdx1b alleviates liver defects in the prox1a mutant zebrafish. Finally, chromatin immunoprecipitation analysis reveals that Prox1a binds directly to the promoter region of cdx1b, thereby repressing its expression. Overall, our findings indicate that Prox1a is required to promote and protect hepatic development by repression of Cdx1b-mediated intestinal cell fate in zebrafish.

FOXC1 and FOXC2 Ablation Causes Abnormal Valvular Endothelial Cell Junctions and Lymphatic Vessel Formation in Myxomatous Mitral Valve Degeneration.

Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2, are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix). Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC-Foxc-DKO and lymphatic EC-Foxc-DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection. EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC-Foxc-DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC-Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC-Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC-Foxc1/c2 mutant MVs. Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration.

ERG Regulates Lymphatic Vessel Specification Genes and Its Deficiency Impairs Wound Healing-Associated Lymphangiogenesis.

Rarefaction of blood and lymphatic vessels in the skin has been reported in systemic sclerosis (SSc) (scleroderma). E26 transformation-specific-related factor (ERG) and Friend leukemia virus-induced erythroleukemia 1 (FLI-1) are important regulators of angiogenesis, but their role in lymphatic vasculature is lesser known. The goal of this study was to determine the role of ERG and FLI-1 in postnatal lymphangiogenesis and SSc lymphatic system defects. Immunofluorescence was used to detect ERG and FLI-1 in skin biopsy samples from patients with SSc and healthy controls. Transcriptional analysis of ERG or FLI-1-silenced human dermal lymphatic endothelial cells (LECs) was performed using microarrays. Effects of ERG and FLI-1 deficiency on in vitro tubulogenesis in human dermal LECs were examined using a Matrigel assay. ERG and FLI-1 endothelial-specific knockouts and ERG lymphatic-specific knockouts were generated to examine vessel regeneration in mice. ERG and FLI-1 protein levels were reduced in the blood and lymphatic vasculature in SSc skin biopsy samples. ERG levels were shown to regulate genes involved in lymphatic vessel specification, including vascular endothelial growth factor receptor 3/FLT-4, lymphatic vessel endothelial hyaluronan receptor 1, SOX-18, and prospero homeobox 1 (PROX-1), whereas FLI-1 enhanced the function of ERG. The ERG-FLT-4 pathway regulated in vitro tubulogenesis in human LECs. Deficiency of ERG or FLI-1 similarly impaired the function of blood vessels in mice. However, only ERG deficiency affected the regeneration of lymphatic vessels during wound healing. ERG and FLI-1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI-1 in SSc endothelial cells may contribute to the impairment of blood and lymphatic vasculature in patients with SSc.

PROX1 is a regulator of neuroendocrine-related gene expression in lung carcinoid.

Lung neuroendocrine neoplasms (NENs) are a diverse group of tumors characterized by neuroendocrine (NE) differentiation. Among lung NENs, lung carcinoid (LC) is a rare tumor with unique characteristics. Recent research has highlighted the importance of transcription factors (TFs) in establishing gene expression programs in lung NENs such as small cell lung carcinoma. However, the TFs that control the gene expression of LC are largely unknown. In this study, we report the expression and potential function of a TF called Prospero homeobox protein1 (PROX1) in LC. Publicly available transcriptome data suggested that PROX1 was highly expressed in LC tissues, which was confirmed by immunohistochemical analysis on a tissue microarray. Knockdown of PROX1 did not impact the cellular viability of an LC-derived cell line, NCI-H727. Meanwhile, transcriptome analysis revealed that PROX1 knockdown altered the expression of genes involved in NE differentiation. ASCL1, CHGA, CALCA, and LINC00261 were suggested as downstream genes of PROX1. These findings indicate that PROX1 may play an important role in the NE identity of LC by regulating the expression of key target genes.

Gastric emphysema and pneumatosis intestinalis in two common marmosets with duodenal dilation syndrome

BackgroundCommon marmosets (Callithrix jacchus) are widely used as primate experimental models in biomedical research. Duodenal dilation with chronic vomiting in captive common marmosets is a recently described life-threatening syndrome that is problematic for health control. However, the pathogenesis and cause of death are not fully understood.Case presentationWe report two novel necropsy cases in which captive common marmosets were histopathologically diagnosed with gastric emphysema (GE) and pneumatosis intestinalis (PI). Marmoset duodenal dilation syndrome was confirmed in each case by clinical observation of chronic vomiting and by gross necropsy findings showing a dilated, gas-filled and fluid-filled descending duodenum that adhered to the ascending colon. A diagnosis of GE and PI was made on the basis of the bubble-like morphology of the gastric and intestinal mucosa, with histological examination revealing numerous vacuoles diffused throughout the lamina propria mucosae and submucosa. Immunostaining for prospero homeobox 1 and CD31 distinguished gas cysts from blood and lymph vessels. The presence of hepatic portal venous gas in case 1 and possible secondary bacteremia-related septic shock in case 2 were suggested to be acute life-threatening abdominal processes resulting from gastric emphysema and pneumatosis intestinalis.ConclusionsIn both cases, the gross and histopathological findings of gas cysts in the GI tract walls matched the features of human GE and PI. These findings contribute to clarifying the cause of death in captive marmosets that have died of gastrointestinal diseases.

Impact of Prospero Homeobox-1 (PROX-1) οn the Oncogenic Phenotypes of Hepatocellular Carcinoma Cells.

Transcriptional factor prospero homeobox-1 (PROX-1) is crucial for the embryonic development of various organs and cell fate specification. It exhibits either an oncogenic or tumor suppressive activity depending on cancer types. However, the relationship between PROX-1 and hepatocellular carcinoma (HCC) remains obscure. This study was conducted to investigate the effect of PROX-1 on the invasive and oncogenic phenotypes of human HCC cells. The effect of PROX-1 on tumor cell behavior was investigated by using a pcDNA-myc vector and a small interfering RNA in HepG2 and Huh7 human HCC cell lines. Flow cytometry, migration, invasion, proliferation, and tube formation assays were performed. PROX-1 expression in human HCC cells was explored by western blotting. PROX-1 overexpression enhanced tumor cell proliferation and inhibited apoptosis and cell cycle arrest by modulating the activities of caspase-3, PARP, and cyclin-dependent kinase inhibitors, including p21, p27, and p57 in HCC cells. After PROX-1 overexpression, the number of migrating and invading HCC cells significantly increased, and the expression levels of N-cadherin and Snail increased in HCC cells. PROX-1 overexpression enhanced angiogenesis through increased VEGF-A and VEGF-C expression and decreased angiostatin expression. PROX-1 overexpression also increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and forkhead box O1 (FOXO1) in HCC cells. After PROX-1 knockdown, their phosphorylation was reversed. PROX-1 overexpression is associated with the invasive and oncogenic phenotypes of human HCC cells via GSK-3β and FOXO1 phosphorylation.

DGAT2 Plays a Crucial Role to Control ESRRA-PROX1 Transcriptional Network to Maintain Hepatic Mitochondrial Sustainability.

Diacylglycerol O-acyltransferase 2 (DGAT2) synthesizes triacylglycerol (TG) from diacylglycerol; therefore, DGAT2 is considered as a therapeutic target for steatosis. However, the consequence of inhibiting DGAT2 is not fully investigated due to side effects including lethality and lipotoxicity. In this article, we observed the role of DGAT2 in hepatocarcinoma. The role of DGAT2 is analyzed via loss-of-function assay. DGAT2 knockdown (KD) and inhibitor treatment on HepG2 cell line was analyzed. Cumulative analysis of cell metabolism with bioinformatic data were assessed, and further compared with different cohorts of liver cancer patients and non-alcoholic fatty liver disease (NAFLD) patients to elucidate how DGAT2 is regulating cancer metabolism. Mitochondrial function is suppressed in DGAT2 KD HepG2 cell along with the decreased lipid droplets. In the aspect of the cancer, DGAT2 KD upregulates cell proliferation. Analyzing transcriptome of NAFLD and hepatocellular carcinoma (HCC) patients highlights negatively correlating expression patterns of 73 lipid-associated genes including DGAT2. Cancer patients with the lower DGAT2 expression face lower survival rate. DGAT2 KD cell and patients' transcriptome show downregulation in estrogen- related receptor alpha (ESRRA) via integrated system for motif activity response analysis (ISMARA), with increased dimerization with corepressor prospero homeobox 1 (PROX1). DGAT2 sustains the stability of mitochondria in hepatoma via suppressing ESRRA-PROX1 transcriptional network and hinders HCC from shifting towards glycolytic metabolism, which lowers cell proliferation.

Molecular expression of Forkhead Box C2 gene (FOXC2) and Prospero homeobox gene (PROX-1) in oral squamous carcinoma and their correlation with clinicopathological parameters: A prospective cohort study.

Forkhead box C2 gene (FOXC2) acts as an epithelial-mesenchymal transition (EMT) inducer while Prospero homeobox 1 gene (PROX-1) function as a regulator of lymphangiogenesis and angiogenesis in oral squamous cell carcinoma (OSCC). It is presumed that PROX-1 has both tumour-suppressive and oncogenic effects. The main aim of this study is to evaluate the role of PROX-1 and FOXC2 in the invasion and progression of OSCC cases and to correlate their expression with various histopathological parameters. A prospective cohort study was conducted in a total sample size of 52 OSCC tissues and histologically tumour-free margins of 20. mRNA expression and protein levels of FOXC2 and PROX-1 were evaluated using real-time PCR and sandwich enzyme-linked immunosorbent assay techniques. Chi-square analysis and correlation analysis were done. Kaplan-Meier analysis evaluated the survival rate. Mean Ct values of FOXC2 were 1.915 ± 0.519 and PROX-1 was 0.061 ± 0.173. There was a significant 2-fold increase in the FOXC2 expression and a 0.5-fold decrease in the PROX-1 expression in OSCC tissue. Increased levels of FOXC2 protein and decreased levels of PROX-1 with a mean difference of 1.64 ± 0.73 ng/ml and 1.27 ± 0.33 ng/ml were observed in OSCC compared to histologically tumour-free margins. A significant positive correlation was found between the FOXC2 expression and clinicopathological parameters such as staging, perineural invasion (PNI) and lymphovascular invasion (LVI) whereas PROX-1 showed a significant negative correlation with histopathological parameters such as staging, PNI, LVI and tumour staging. There was a significant positive correlation between the PROX-1 and histologically tumour-free margins in disease-free survival patients (P-value = 0.03). FOXC2 and PROX-1 expressions were correlated with lymphovascular invasion, OSCC tumour staging and PNI. Thus, FOXC2 and PROX-1 could be possible therapeutic targets in the treatment of OSCC that can inhibit the EMT in OSCC and thereby favouring a better prognosis.

Knockdown of PROX1 promotes milk fatty acid synthesis by targeting PPARGC1A in dairy goat mammary gland

Goat milk is rich in various fatty acids that are beneficial to human health. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-seq analyses of goat mammary glands at different lactation stages revealed a novel lactation regulatory factor, Prospero homeobox 1 (PROX1). However, the mechanism whereby PROX1 regulates lipid metabolism in dairy goats remains unclear. We found that PROX1 exhibits the highest expression level during peak lactation period. PROX1 knockdown enhanced the expression of genes related to de novo fatty acid synthesis (e.g., SREBP1 and FASN) and triacylglycerol (TAG) synthesis (e.g., DGAT1 and GPAM) in goat mammary epithelial cells (GMECs). Consistently, intracellular TAG and lipid droplet contents were significantly increased in PROX1 knockdown cells and reduced in PROX1 overexpression cells, and we observed similar results in PROX1 knockout mice. Following PROX1 overexpression, RNA-seq showed a significant upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) expression. Further, PPARGC1A knockdown attenuated the inhibitory effects of PROX1 on TAG contents and lipid-droplet formation in GMECs. Moreover, we found that PROX1 promoted PPARGC1A transcription via the PROX1 binding sites (PBSs) located in the PPARGC1A promoter. These results suggest a novel target for manipulating the goat milk-fat composition and improving the quality of goat milk.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1.

Retinoblastoma (RB) is a prevalent type of eye cancer in youngsters. Prospero homeobox 1 (Prox1) is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic, hepatocyte, pancreatic, heart, lens, retinal, and cancer cells. The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance, as well as to explore the underlying Notch1 mechanism. Human RB cell lines (SO-RB50 and Y79) and a primary human retinal microvascular endothelial cell line (ACBRI-181) were used in this study. The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay. Drug-resistant cell lines (SO-RB50/vincristine) were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance. We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1. Finally, a xenograft model was constructed to assess the effect of Prox1 on RB in vivo. Prox1 was significantly downregulated in RB cells. Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine. Notch1 was involved in Prox1's regulatory effects. Notch1 was identified as a target gene of Prox1, which was found to be upregulated in RB cells and repressed by increased Prox1 expression. When pcDNA-Notch1 was transfected, the effect of Prox1 overexpression on RB was removed. Furthermore, by downregulating Notch1, Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo. These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1, implying that Prox1 could be a potential therapeutic target for RB.

Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

PROX1 interaction with α-SMA-rich cancer-associated fibroblasts facilitates colorectal cancer progression and correlates with poor clinical outcomes and therapeutic resistance.

The tumor microenvironment (TME) plays a vital role in tumor progression through intricate molecular interactions. Cancer-associated fibroblasts (CAFs), notably those expressing alpha-smooth muscle actin (α-SMA) or myofibroblasts, are instrumental in this context and correlate with unfavorable outcomes in colorectal cancer (CRC). While several transcription factors influence TME, the exact regulator causing CAF dysregulation in CRC remains elusive. Prospero Homeobox 1 (PROX1) stands out, as its inhibition reduces α-SMA-rich CAF activity. However, the therapeutic role of PROX1 is debated due to inconsistent study findings. Using the ULCAN portal, we noted an elevated PROX1 level in advanced colon adenocarcinoma, linking to a poor prognosis. Assays determined the impact of PROX1 overexpression on CRC cell properties, while co-culture experiments spotlighted the PROX1-CAF relationship. Molecular expressions were validated by qRT-PCR and Western blots, with in vivo studies further solidifying the observations. Our study emphasized the connection between PROX1 and α-SMA in CAFs. Elevated PROX1 in CRC samples correlated with increased α-SMA in tumors. PROX1 modulation influenced the behavior of specific CRC cells, with its overexpression fostering invasiveness. Kaplan-Meier evaluations demonstrated a link between PROX1 or α-SMA and survival outcomes. Consequently, PROX1, alone or with α-SMA, emerges as a CRC prognostic marker. Co-culture and animal experiments further highlighted this relationship. PROX1 appears crucial in modulating CRC behavior and therapeutic resistance within the TME by influencing CAFs, signifying the combined PROX1/α-SMA gene as a potential CRC prognostic marker. The concept of developing inhibitors targeting this gene set emerges as a prospective therapeutic strategy. However, this study is bound by limitations, including potential challenges in clinical translation, a focused exploration on PROX1/α-SMA potentially overlooking other significant molecular contributors, and the preliminary nature of the inhibitor development proposition.

Comparative Analysis of Vascular Cell Differentiation From Peripheral Blood Mononuclear Cell‐ and Urine‐Derived Induced Pluripotent Stem Cells

Background and Objectives: Peripheral blood mononuclear cells (PBMCs) and urine‐derived epithelial cells have both emerged as valuable sources for induced pluripotent stem cell (iPSC) generation, each presenting unique advantages in terms of accessibility and reprograming efficiency. This study aimed to assess and compare the potential of PBMC‐derived iPSCs (PiPSCs) and urine‐derived iPSCs (UiPSCs) in generating functional endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), which are critical for vascular tissue engineering and disease modeling. Phenotypic characteristics, differentiation efficacy, and functional properties of iPSC‐derived ECs and VSMCs from these distinct sources were investigated to reveal variations attributed to cellular origin.Methods and Results: PiPSCs and UiPSCs both successfully differentiated into functional ECs and VSMCs. EC differentiation efficiency was similar, yielding about 45% mature ECs with characteristic morphology, marker expression, and tube formation abilities, showing no significant differences between cell types. Transcriptomic analysis revealed upregulation of key endothelial markers (platelet endothelial cell adhesion molecule 1 [PECAM1], cadherin 5 [CDH5], and melanoma cell adhesion molecule [MCAM]) and downregulation of lymphatic markers (Fms‐related tyrosine kinase 4 [FLT4], prospero homeobox protein 1 [PROX1], and podoplanin [PDPN]), confirming blood EC identity. The upregulation of collagen type IV alpha 1 chain (COL4A1) and collagen type I alpha 1 chain (COL1A1) indicated a mature endothelial state with enhanced extracellular matrix (ECM) production. VSMC differentiation resulted in high percentages of α‐smooth muscle actin (α‐SMA) positive cells for both PiPSCs (96%) and UiPSCs (94%). These VSMCs exhibited typical spindle‐shaped morphology, expressed VSMC markers, and responded to carbachol. Transcriptomic analysis showed significant upregulation of VSMC markers (actin alpha 2 [ACTA2], caldesmon 1 [CALD1], calponin 1 [CNN1], transgelin [TAGLN], tropomyosin 2 [TPM2]), with concurrent downregulation of ACTA1 and upregulation of ACTA2, confirming their vascular smooth muscle phenotype. The upregulation of COL6A1 in VSMCs indicated a mature phenotype with enhanced ECM production, crucial for vascular tissue integrity and function. Gene set enrichment analysis highlighted the upregulation of multiple hallmark pathways, delineating a distinctive transcriptional profile.Conclusions: This study presents a comprehensive comparative analysis of functionally differentiated ECs and VSMCs derived from PiPSCs and UiPSCs, providing critical insights into the expression patterns and phenotypic transitions during differentiation. Our findings enhance the understanding of distinct molecular signatures in iPSCs from different sources and their progeny.

Electroacupuncture promotes neurogenesis in the dentate gyrus and improves pattern separation in an early Alzheimer's disease mouse model

BackgroundImpaired pattern separation occurs in the early stage of Alzheimer’s disease (AD), and hippocampal dentate gyrus (DG) neurogenesis participates in pattern separation. Here, we investigated whether spatial memory discrimination impairment can be improved by promoting the hippocampal DG granule cell neogenesis-mediated pattern separation in the early stage of AD by electroacupuncture (EA).MethodsFive familial AD mutations (5 × FAD) mice received EA treatment at Baihui and Shenting points for 4 weeks. During EA, mice were intraperitoneally injected with BrdU (50 mg/kg) twice a day. rAAV containing Wnt5a shRNA was injected into the bilateral DG region, and the viral efficiency was evaluated by detecting Wnt5a mRNA levels. Cognitive behavior tests were conducted to assess the impact of EA treatment on cognitive function. The hippocampal DG area Aβ deposition level was detected by immunohistochemistry after the intervention; The number of BrdU+/CaR+ cells and the gene expression level of calretinin (CaR) and prospero homeobox 1(Prox1) in the DG area of the hippocampus was detected to assess neurogenesis by immunofluorescence and western blotting after the intervention; The gene expression levels of FZD2, Wnt5a, DVL2, p-DVL2, CaMKII, and p-CaMKII in the Wnt signaling pathway were detected by Western blotting after the intervention.ResultsCognitive behavioral tests showed that 5 × FAD mice had impaired pattern separation (P < 0.001), which could be improved by EA (P < 0.01). Immunofluorescence and Western blot showed that the expression of Wnt5a in the hippocampus was decreased (P < 0.001), and the neurogenesis in the DG was impaired (P < 0.001) in 5 × FAD mice. EA could increase the expression level of Wnt5a (P < 0.05) and promote the neurogenesis of immature granule cells (P < 0.05) and the development of neuronal dendritic spines (P < 0.05). Interference of Wnt5a expression aggravated the damage of neurogenesis (P < 0.05), weakened the memory discrimination ability (P < 0.05), and inhibited the beneficial effect of EA (P < 0.05) in AD mice. The expression level of Wnt pathway related proteins such as FZD2, DVL2, p-DVL2, CAMKII, p-CAMKII increased after EA, but the effect of EA was inhibited after Wnt5a was knocked down. In addition, EA could reduce the deposition of Aβ plaques in the DG without any impact on Wnt5a.ConclusionEA can promote hippocampal DG immature granule cell neogenesis-mediated pattern separation to improve spatial memory discrimination impairment by regulating Wnt5a in 5 × FAD mice.

Her6 and Prox1a are novel regulators of photoreceptor regeneration in the zebrafish retina

Damage to light-sensing photoreceptors (PRs) occurs in highly prevalent retinal diseases. As humans cannot regenerate new PRs, these diseases often lead to irreversible blindness. Intriguingly, animals, such as the zebrafish, can regenerate PRs efficiently and restore functional vision. Upon injury, mature Müller glia (MG) undergo reprogramming to adopt a stem cell-like state. This process is similar to cellular dedifferentiation, and results in the generation of progenitor cells, which, in turn, proliferate and differentiate to replace lost retinal neurons. In this study, we tested whether factors involved in dedifferentiation of Drosophila CNS are implicated in the regenerative response in the zebrafish retina. We found that hairy-related 6 (her6) negatively regulates of PR production by regulating the rate of cell divisions in the MG-derived progenitors. prospero homeobox 1a (prox1a) is expressed in differentiated PRs and may promote PR differentiation through phase separation. Interestingly, upon Her6 downregulation, Prox1a is precociously upregulated in the PRs, to promote PR differentiation; conversely, loss of Prox1a also induces a downregulation of Her6. Together, we identified two novel candidates of PR regeneration that cross regulate each other; these may be exploited to promote human retinal regeneration and vision recovery.

Prospero Homeobox 1 and Doublecortin Correlate with Neural Damage after Ischemic Stroke.

Markers of neuroinflammation during ischemic stroke are well characterized, but additional markers of neural damage are lacking. The study identified associations of behavioral disorders after stroke with histologic neural damage and molecular biological change. Eight-week-old, 25 g male mice of the C57BL/6J strain were subjected to middle cerebral artery occlusion (MCAO) to induce ischemic stroke. The control group was a healthy wild type (WT), and the experimental group were designed as a low severity MCAO1 and a high severity MCAO2 based on post-stroke neurological scoring. All groups underwent behavioral tests, realtime polymerase chain reaction, triphenyltetrazolium chloride (TTC) staining and Hematoxylin and Eosin staining. One-way analysis of variance was used to analyze statistical significance between groups. In TTC staining, MCAO1 showed 29.02% and MCAO2 showed 38.94% infarct volume (p<0.0001). The pro-inflammatory cytokine interleukin (IL)-1β was most highly expressed in MCAO2 (WT 0.44 vs. MCAO1 2.69 vs. MCAO2 5.02, p<0.0001). From the distance to target in the Barnes maze test, WT had a distance of 178 cm, MCAO1 had a distance of 276 cm, and MCAO2 had a distance of 1051 (p=0.0015). The latency to target was 13.3 seconds for WT, 27.9 seconds for MCAO1, and 87.9 seconds for MCAO2 (p=0.0007). Prospero homeobox 1 (Prox1) was most highly expressed in MCAO2 (p=0.0004). Doublecortin (Dcx) was most highly expressed in MCAO2 (p<0.0001). The study demonstrated that histological damage to neural cells and changes in brain mRNA expression were associated with behavioral impairment after ischemic stroke. Prox1 and Dcx may be biomarkers of neural damage associated with long-term cognitive decline, and increased expression at the mRNA level was consistent with neural damage and long-term cognitive dysfunction.

Abstract B001: Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment

Abstract Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive cancer. In the UK, there are around 9000 diagnoses and 3000 deaths annually. Targeted therapies that inhibit angiogenic regulators, such as bevacizumab (monoclonal antibody which inhibits vascular endothelial growth factor A, VEGF-A), have limited efficacy due to drug resistance. Expression of calcitonin receptor-like receptor (CLR) is upregulated in ccRCC and correlates with disease outcome. The expression of adrenomedullin (AM), peptide agonist of CLR and angiogenic factor, is upregulated in ccRCC. We hypothesized that the CLR signaling axis acts as an alternative pathway that enables resistance to drugs targeting the VEGF-A pathway in ccRCC. In the present study, we aimed to determine whether this signaling axis remains active in VEGF-A-stimulated and bevacizumab-treated human endothelial cells. Methods. Primary human dermal blood vessel endothelial cells (HDBEC; PromoCell) were characterized by immunofluorescence utilizing pan-endothelial (cluster of differentiation 31 and 144, CD31 and CD144, and von Willebrand factor, vWF) and lymphatic-specific (lymphatic vessel endothelial hyaluronan receptor 1, LYVE-1, and prospero homeobox 1, PROX1) markers and confocal microscopy. The activities of CLR agonists (AM; intermedin, IMD; calcitonin gene-related peptide, CGRP; 10−12 - 10−6 M at 10 min) and VEGF-A (50 ng/ml over 30-minute time course) were measured by analyzing phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) using immunoblotting. HDBEC were pre-treated with bevacizumab or human IgG1 at 250 ng/ml and then stimulated with individual CLR agonists at 10−6 M for 10 minutes. Experiments were repeated at least three times. Data were analyzed using Shapiro-Wilk normality, unpaired statistics and appropriate multiple comparisons tests utilizing GraphPad Prism and p&amp;lt;0.05 interpreted as significant. Results. HDBEC were a pure population. AM, IMD and CGRP induced p44/42 MAPK phosphorylation at 10−6 M (p&amp;lt;0.05). For all three CLR agonists and VEGF-A, p44/42 MAPK phosphorylation peaked after 10 minutes of incubation (p&amp;lt;0.05). Bevacizumab treatment at 250 ng/ml was sufficient to inhibit VEGF-A-induced p44/42 MAPK phosphorylation (p&amp;lt;0.05). AM, IMD and CGRP were all able to induce phosphorylation of p44/42 MAPK after bevacizumab treatment (p&amp;lt;0.01). Conclusions. The findings that CLR agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated HDBEC after bevacizumab treatment support our hypothesis. This work provides a foundation for investigating the role of the CLR signaling axis in mechanisms of ccRCC resistance to drugs targeting the VEGF-A pathway. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Leonid L. Nikitenko. Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B001.

The use of commercial fibrin glue in dermal replacement material reduces angiogenic and lymphangiogenic gene and protein expression in vitro.

Commercial fibrin glue is increasingly finding its way into clinical practice in surgeries to seal anastomosis, and initiate hemostasis or tissue repair. Human biological glue is also being discussed as a possible cell carrier. To date, there are only a few studies addressing the effects of fibrin glue on the cell-molecular level. This study examines the effects of fibrin glue on angiogenesis and lymphangiogenesis, as well as adipose-derived stem cells (ASCs) with a focus on gene and protein expression in scaffolds regularly used for tissue engineering approaches. Collagen-based dermal regeneration matrices (DRM) were seeded with human umbilical vein endothelial cells (HUVEC), human dermal lymphatic endothelial cells (LECs), or adipose-derived stem cells (ASC) and fixed with or without fibrin glue according to the experimental group. Cultures were maintained for 1 and 7days. Finally, angiogenic and lymphangiogenic gene and protein expression were measured with special regard to subtypes of vascular endothelial growth factor (VEGF) and corresponding receptors using Multiplex-qPCR and ELISA assays. In addition, the hypoxia-induced factor 1-alpha (HIF1a) mediated intracellular signaling pathways were included in assessments to analyze a hypoxic encapsulating effect of fibrin polymers. All cell types reacted to fibrin glue application with an alteration of gene and protein expression. In particular, vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor C (VEGFC), vascular endothelial growth receptor 1 (VEGFR1/FLT1), vascular endothelial growth receptor 2 (VEGFR2/KDR), vascular endothelial growth receptor 3 (VEGFR3/FLT4) and Prospero Homeobox 1 (PROX1) were depressed significantly depending on fibrin glue. Especially short-term fibrin effect led to a continuous downregulation of respective gene and protein expression in HUVECs, LECs, and ASCs. Our findings demonstrate the impact of fibrin glue application in dermal regeneration with special regard to angiogenesis and lymphangiogenesis. In particular, a short fibrin treatment of 24hours led to a decrease in gene and protein levels of LECS, HUVECs, and ASCs. In contrast, the long-term application showed less effect on gene and protein expressions. Therefore, this work demonstrated the negative effects of fibrin-treated cells in tissue engineering approaches and could affect wound healing during dermal regeneration.

Adipose-Derived Stem Cells Improve Angiogenesis and Lymphangiogenesis in a Hypoxic Dermal Regeneration Model In Vitro.

Background and Objectives: Impaired wound healing represents an unsolved medical issue with a high impact on patients' quality of life and global health care. Even though hypoxia is a significant limiting factor for wound healing, it reveals stimulating effects in gene and protein expression at cellular levels. In particular, hypoxically treated human adipose tissue-derived stem cells (ASCs) have previously been used to stimulate tissue regeneration. Therefore, we hypothesized that they could promote lymphangiogenesis or angiogenesis. Materials and Methods: Dermal regeneration matrices were seeded with human umbilical vein endothelial cells (HUVECs) or human dermal lymphatic endothelial cells (LECs) that were merged with ASCs. Cultures were maintained for 24 h and 7 days under normoxic or hypoxic conditions. Finally, gene and protein expression were measured regarding subtypes of VEGF, corresponding receptors, and intracellular signaling pathways, especially hypoxia-inducible factor-mediated pathways using multiplex-RT-qPCR and ELISA assays. Results: All cell types reacted to hypoxia with an alteration of gene expression. In particular, vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor C (VEGFC), vascular endothelial growth factor receptor 1 (VEGFR1/FLT1), vascular endothelial growth factor receptor 2 (VEGFR2/KDR), vascular endothelial growth factor receptor 3 (VEGFR3/FLT4), and prospero homeobox 1 (PROX1) were overexpressed significantly depending on upregulation of hypoxia-inducible factor 1 alpha (HIF-1a). Moreover, co-cultures with ASCs showed a more intense change in gene and protein expression profiles and gained enhanced angiogenic and lymphangiogenic potential. In particular, long-term hypoxia led to continuous stimulation of HUVECs by ASCs. Conclusions: Our findings demonstrated the benefit of hypoxic conditioned ASCs in dermal regeneration concerning angiogenesis and lymphangiogenesis. Even a short hypoxic treatment of 24 h led to the stimulation of LECs and HUVECs in an ASC-co-culture. Long-term hypoxia showed a continuous influence on gene expressions. Therefore, this work emphasizes the supporting effects of hypoxia-conditioned-ASC-loaded collagen scaffolds on wound healing in dermal regeneration.