Urotensin II receptor deficiency ameliorates ligation-induced carotid intimal hyperplasia partially through the RhoA-YAP1 pathway.

Intimal hyperplasia (IH) is a common pathological feature of vascular proliferative diseases, such as atherosclerosis and restenosis after angioplasty. Urotensin II (UII) and its receptor (UTR) are widely expressed in cardiovascular tissues. However, it remains unclear whether the UII/UTR system is involved in IH. Right unilateral common carotid artery ligation was performed and maintained for 21 days to induce IH in UTR knockout (UTR-/-) and wild-type (WT) mice. Histological analysis revealed that compared with WT mice, UTR-deficient mice exhibited a decreased neointimal area, angiostenosis and intima-media ratio. Immunostaining revealed fewer smooth muscle cells (SMCs), endothelial cells and macrophages in the lesions of UTR-/- mice than in those of WT mice. Protein interaction analysis suggested that the UTR may affect cell proliferation by regulating YAP and its downstream target genes. In vitro experiments revealed that UII can promote the proliferation and migration of SMCs, and western blotting also revealed that UII increased the protein expression of RhoA, CTGF, Cyclin D1 and PCNA and downregulated p-YAP protein expression, while these effects could be partly reversed by urantide. To evaluate the translational value of UTRs in IH management, WT mice were also treated with two doses of urantide, a UTR antagonist, to confirm the benefit of UTR blockade in IH progression. A high dose of urantide (600 µg/kg/day), rather than a low dose (60 µg/kg/day), successfully improved ligation-induced IH compared with that in mice receiving vehicle. The results of the present study suggested that the UII/UTR system may regulate IH partly through the RhoA-YAP signaling pathway.

Endothelial POFUT1 controls injury-induced liver fibrosis by repressing fibrinogen synthesis.

BACKGROUND & AIMS: NOTCH signaling in liver sinusoidal endothelial cells (LSECs) regulates liver fibrosis, a pathological feature of chronic liver diseases. POFUT1 is an essential regulator of NOTCH signaling. Here, we investigated the role of LSEC-expressed POFUT1 in liver fibrosis. METHODS: Endothelial-specific Pofut1 knockout mice were generated and experimental liver fibrosis was induced by chronic carbon tetrachloride exposure or common bile duct ligation. Liver samples were assessed by ELISA, histology, electron microscopy, immunostaining and RNA in situ hybridization. LSECs and hepatic stellate cells (HSCs) were isolated for gene expression analysis by RNA sequencing, qPCR, and western blotting. Signaling crosstalk between LSECs and HSCs was investigated by treating HSCs with supernatant from LSEC cultures. Liver single-cell RNA sequencing datasets from patients with cirrhosis and healthy individuals were analyzed to evaluate the clinical relevance of gene expression changes observed in mouse studies. RESULTS: POFUT1 loss promoted injury-induced LSEC capillarization and HSC activation, leading to aggravated liver fibrosis. RNA sequencing analysis revealed that POFUT1 deficiency upregulated fibrinogen expression in LSECs. Consistently, fibrinogen was elevated in LSECs of patients with cirrhosis. HSCs treated with supernatant from LSECs of Pofut1 null mice showed exacerbated activation compared to those treated with supernatant from control LSECs, and this effect was attenuated by knockdown of fibrinogen or by pharmacological inhibition of fibrinogen receptor signaling, altogether suggesting that LSEC-derived fibrinogen induced the activation of HSCs. Mechanistically, POFUT1 loss augmented fibrinogen expression by enhancing NOTCH/HES1/STAT3 signaling. CONCLUSIONS: Endothelial POFUT1 prevents injury-induced liver fibrosis by repressing the expression of fibrinogen, which functions as a profibrotic paracrine signal to activate HSCs. Therapies targeting the POFUT1/fibrinogen axis offer a promising strategy for the prevention and treatment of fibrotic liver diseases. IMPACT AND IMPLICATIONS: Paracrine signals produced by liver vasculature play a major role in the development of liver fibrosis, which is a pathological hallmark of most liver diseases. Identifying those paracrine signals is clinically relevant in that they may serve as therapeutic targets. In this study, we discovered that genetic deletion of Pofut1 aggravated experimental liver fibrosis in mouse models. Moreover, fibrinogen was identified as a downstream target repressed by Pofut1 in liver endothelial cells and functioned as a novel paracrine signal that drove liver fibrosis. In addition, fibrinogen was found to be relevant to cirrhosis and may serve as a potential therapeutic target for this devastating human disease.

Dysfunction of DMT1 and miR-135b in the gut-testis axis in high-fat diet male mice.

BACKGROUND: Obese patients have been found to be susceptible to iron deficiency, and malabsorption of dietary iron is the cause of obesity-related iron deficiency (ORID). Divalent metal transporter 1 (DMT1) and ferroportin (FPN), are two transmembrane transporter proteins expressed in the duodenum that are closely associated with iron absorption. However, there have been few studies on the association between these two proteins and the increased susceptibility to iron deficiency in obese patients. Chronic inflammation is also thought to be a cause of obesity-related iron deficiency, and both conditions can have an impact on spermatogenesis and impair male reproductive function. Based on previous studies, transgenerational epigenetic inheritance through gametes was observed in obesity. RESULTS: Our results  showed that obese mice had decreased blood iron levels (p < 0.01), lower protein and mRNA expression for duodenal DMT1 (p < 0.05), but no statistically significant variation in mRNA expression for duodenal FPN (p > 0.05); there was an increase in sperm miR-135b expression (p < 0.05). Bioinformatics revealed ninety overlapping genes and further analysis showed that they were primarily responsible for epithelial cilium movement, fatty acid beta-oxidation, protein dephosphorylation, fertilization, and glutamine transport, which are closely related to spermatogenesis, sperm development, and sperm viability in mice. CONCLUSIONS: In obese mice, we observed downregulation of DMT1 in the duodenum and upregulation of miR-135b in the spermatozoa.

Paternal high-fat diet altered H3K36me3 pattern of pre-implantation embryos.

The global transition towards diets high in calories has contributed to 2.1 billion people becoming overweight, or obese, which damages male reproduction and harms offspring. Recently, more and more studies have shown that paternal exposure to stress closely affects the health of offspring in an intergenerational and transgenerational way. SET Domain Containing 2 (SETD2), a key epigenetic gene, is highly conserved among species, is a crucial methyltransferase for converting histone 3 lysine 36 dimethylation (H3K36me2) into histone 3 lysine 36 trimethylation (H3K36me3), and plays an important regulator in the response to stress. In this study, we compared patterns of SETD2 expression and the H3K36me3 pattern in pre-implantation embryos derived from normal or obese mice induced by high diet. The results showed that SETD2 mRNA was significantly higher in the high-fat diet (HFD) group than the control diet (CD) group at the 2-cell, 4-cell, 8-cell, and 16-cell stages, and at the morula and blastocyst stages. The relative levels of H3K36me3 in the HFD group at the 2-cell, 4-cell, 8-cell, 16-cell, morula stage, and blastocyst stage were significantly higher than in the CD group. These results indicated that dietary changes in parental generation (F0) male mice fed a HFD were traceable in SETD2/H3K36me3 in embryos, and that a paternal high-fat diet brings about adverse effects for offspring that might be related to SETD2/H3K36me3, which throws new light on the effect of paternal obesity on offspring from an epigenetic perspective.

Significant alteration of protein profiles in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder, affecting women of child-bearing age, and the incidence rate is growing and assuming epidemic proportions. The etiology of PCOS remains unknown and there is no cure. Some animal models for PCOS have been established which have enhanced our understanding of the underlying mechanisms, but omics data for revealing PCOS pathogenesis and for drug discovery are still lacking. In the present study, proteomics analysis was used to construct a protein profile of the ovaries in a PCOS mouse model. The result showed a clear difference in protein profile between the PCOS and control group, with 495 upregulated proteins and 404 downregulated proteins in the PCOS group. The GO term and KEGG pathway analyses of differentially expressed proteins mainly showed involvement in lipid metabolism, oxidative stress, and immune response, which are consistent with pathological characteristics of PCOS in terms of abnormal metabolism, endocrine disorders, chronic inflammation and imbalance between oxidant and antioxidant levels. Also, we found that inflammatory responses were activated in the PCOS ovarium, while lipid biosynthetic process peroxisome, and bile secretion were inhibited. In addition, we found some alteration in unexpected pathways, such as glyoxylate and dicarboxylate metabolism, which should be investigated. The present study makes an important contribution to the current lack of PCOS ovarian proteomic data and provides an important reference for research and development of effective drugs and treatments for PCOS.

The incidence rate and histological characteristics of intimal hyperplasia in elastase-induced experimental abdominal aortic aneurysms in mice.

Intimal hyperplasia (IH) is a negative vascular remodeling after arterial injury. IH occasionally occurs in elastase-induced abdominal aortic aneurysm (AAA) mouse models. This study aims to clarify the incidence and histological characteristics of IH in aneurysmal mice. A retrospective study was conducted by including 42 male elastase-induced mouse AAA models. The IH incidence, aortic diameters with or without IH, and hyperplasia lesional features of mice were analyzed. Among 42 elastase-induced AAA mouse models, 10 mice developed mild IH (24%) and severe IH was found in only 2 mice (5%). The outer diameters of the AAA segments in mice with and without IH did not show significant difference. Both mild and severe IH lesions show strong smooth muscle cell positive staining, but endothelial cells were occasionally observed in severe IH lesions. There was obvious macrophage infiltration in the IH lesions of the AAA mouse models, especially in mice with severe IH. However, only a lower numbers of T cells and B cells were found in the IH lesion. Local cell-secreted matrix metalloproteinases (MMP) 2 was highly expressed in all IH lesions, but MMP9 was only overexpressed in severe lesions. In conclusion, this study is the first to demonstrate the occurrence of aneurysmal IH and its histological characteristics in an elastase-induced mouse AAA model. This will help researchers better understand this model, and optimize it for use in AAA-related research.

C-reactive protein deficiency ameliorates experimental abdominal aortic aneurysms.

Background: C-reactive protein (CRP) levels are elevated in patients with abdominal aortic aneurysms (AAA). However, it has not been investigated whether CRP contributes to AAA pathogenesis. Methods: CRP deficient and wild type (WT) male mice were subjected to AAA induction via transient intra-aortic infusion of porcine pancreatic elastase. AAAs were monitored by in situ measurements of maximal infrarenal aortic external diameters immediately prior to and 14 days following elastase infusion. Key AAA pathologies were assessed by histochemical and immunohistochemical staining procedures. The influence of CRP deficiency on macrophage activation was evaluated in peritoneal macrophages in vitro. Results: CRP protein levels were higher in aneurysmal than that in non-aneurysmal aortas. Aneurysmal aortic dilation was markedly suppressed in CRP deficient (aortic diameter: 1.08 ± 0.11 mm) as compared to WT (1.21 ± 0.08 mm) mice on day 14 after elastase infusion. More medial elastin was retained in CRP deficient than in WT elastase-infused mice. Macrophage accumulation was significantly less in aneurysmal aorta from CRP deficient than that from WT mice. Matrix metalloproteinase 2 expression was also attenuated in CRP deficient as compared to WT aneurysmal aortas. CRP deficiency had no recognizable influence on medial smooth muscle loss, lymphocyte accumulation, aneurysmal angiogenesis, and matrix metalloproteinase 9 expression. In in vitro assays, mRNA levels for tumor necrosis factor α and cyclooxygenase 2 were reduced in lipopolysaccharide activated peritoneal macrophages from CRP deficient as compared to wild type mice. Conclusion: CRP deficiency suppressed experimental AAAs by attenuating aneurysmal elastin destruction, macrophage accumulation and matrix metalloproteinase 2 expression.

Mediator subunit MED1 deficiency prevents carbon tetrachloride-induced hepatic fibrosis in mice.

Mediator subunit mediator 1 (MED1) mediates ligand-dependent binding of the mediator coactivator complex to various nuclear receptors and plays a critical role in embryonic development, lipid and glucose metabolism, liver regeneration, and tumorigenesis. However, the precise role of MED1 in the development of liver fibrosis has been unclear. Here, we showed that MED1 expression was increased in livers from nonalcoholic steatohepatitis (NASH) patients and mice and positively correlated with transforming growth factor ß (TGF-ß) signaling and profibrotic factors. Upon treatment with carbon tetrachloride (CCl4), hepatic fibrosis was much less in liver-specific MED1 deletion (MED1ΔLiv) mice than in MED1fl/fl littermates. TGF-ß/Smad2/3 signaling pathway was inhibited, and gene expression of fibrotic markers, including α-smooth muscle actin (α-SMA), collagen type 1 α 1 (Col1a1), matrix metalloproteinase-2 (Mmp2), and metallopeptidase inhibitor 1 (Timp1) were decreased in livers of MED1ΔLiv mice with CCl4 injection. Transcriptomic analysis revealed that the differentially expressed genes in livers of CCl4-administered MED1ΔLiv mice were enriched in the pathway of oxidoreductase activity, followed by robustly reduced oxidoreductase activity-related genes, such as Gm4756, Txnrd3, and Etfbkmt. More importantly, we found that the reduction of reactive oxygen species (ROS) in MED1 knockdown hepatocytes blocked the activation of TGF-ß/Smad2/3 pathway and the expression of fibrotic genes in LX2 cells. These results indicate that MED1 is a positive regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for the regression of liver fibrosis.NEW & NOTEWORTHY In this study, we present the first evidence that liver mediator 1 (MED1) deficiency attenuated carbon tetrachloride-induced hepatic fibrosis in mouse. The underlying mechanism is that MED1 deficiency reduces reactive oxygen species (ROS) production in hepatocytes, thus restricts the activation of TGF-ß/Smad2/3 signaling pathway and fibrogenic genes expression in hepatic stellate cells (HSCs). These data suggest that MED1 is an essential regulator for hepatic fibrogenesis, and MED1 may be considered as a potential therapeutic target for liver fibrosis.

Hepatic transcriptome signatures in mice and humans with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the main reason for cirrhosis and hepatocellular carcinoma. As a starting point for NAFLD, the treatment of nonalcoholic fatty liver (NAFL) is receiving increasing attention. Mice fed a high-fat diet (HFD) and hereditary leptin deficiency (ob/ob) mice are important NAFL animal models. However, the comparison of these mouse models with human NAFL is still unclear. METHODS: In this study, HFD-fed mice and ob/ob mice were used as NAFL animal models. Liver histopathological characteristics were compared, and liver transcriptome from both mouse models was performed using RNA sequencing (RNA-seq). RNA-seq data obtained from the livers of NAFL patients was downloaded from the GEO database. Global gene expression profiles in the livers were further analyzed using functional enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. RESULTS: Our results showed that the biochemical parameters of both mouse models and human NAFL were similar. Compared with HFD-fed mice, ob/ob mice were more similar in histologic appearance to NAFL patients. The liver transcriptome characteristics partly overlapped in mice and humans. Furthermore, in the NAFL pathway, most genes showed similar trends in mice and humans, thus demonstrating that both types of mice can be used as models for basic research on NAFL, considering the differences. CONCLUSION: Our findings show that HFD-fed mice and ob/ob mice can mimic human NAFL partly in pathophysiological process. The comparative analysis of liver transcriptome profile in mouse models and human NAFL presented here provides insights into the molecular characteristics across these NAFL models.

Deficiency of protein inhibitor of activated STAT3 exacerbates atherosclerosis by modulating VSMC phenotypic switching.

BACKGROUND AND AIMS: Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an essential role in the development of atherosclerosis. Protein inhibitor of activated STAT (Pias) regulates VSMCs phenotype via acting as sumo E3 ligase to promote protein sumoylation. Our previous study indicated that Pias3 expression decreased in atherosclerotic lesions. Therefore, this study aimed to explore the role of Pias3 on VSMCs phenotype switching during atherosclerosis. METHODS: ApoE-/- and ApoE-/-Pias3-/- double-deficient mice were fed with high-fat/high-cholesterol diet to induce atherosclerosis. Aorta tissues and primary VSMCs were collected to assess plaque formation and VSMCs phenotype. In vitro, Pias3 was overexpressed in A7r5, a VSMCs cell line, by transfection with Pias3 plasmid. Real-time quantitative PCR, immunoblotting, immunoprecipitation, were used to analyze the effect of Pias3 on VSMCs phenotypic switching. RESULTS: Pias3 deficiency significantly exacerbated atherosclerotic plaque formation and promoted VSMCs phenotypic switching to a synthetic state within lesion. In vitro, overexpressing Pias3 in VSMCs increased the expression of contractile markers (myosin heavy chain 11, calponin 1), while it decreased the level of synthetic marker (vimentin). Additionally, Pias3 overexpression blocked PDGF-BB-induced VSMCs proliferation and migration. Immunoprecipitation and mass spectrometry results showed that Pias3 enhanced sumoylation and ubiquitination of vimentin, and shortened its half-life. Moreover, the ubiquitination level of vimentin was impaired by 2-D08, a sumoylation inhibitor. This suggests that Pias3 might accelerate the ubiquitination-degradation of vimentin by promoting its sumoylation. CONCLUSIONS: These results indicate that Pias3 might ameliorate atherosclerosis progression by suppressing VSMCs phenotypic switching and reducing vimentin protein stability.

A homozygous missense mutation in the fibroblast growth factor 5 gene is associated with the long-hair trait in Angora rabbits.

BACKGROUND: Rabbits are well-domesticated animals. As a crucial economic animal, rabbit has been successfully bred into wool-use, meat-use and fur-use breeds. Hair length is one of the most economically important traits affecting profitability in wool rabbits. In this study, to identify selection signatures with the long-hair trait, whole-genomic resequencing of long-haired rabbits (Angora rabbits) and short-haired rabbits (Rex and New Zealand rabbits) was performed. RESULTS: By genome-wide selective sweeping analysis based on population comparison, we identified a total of 5.85 Mb regions (containing 174 candidate genes) with strong selection signals. Six of these genes (Dusp1, Ihh, Fam134a, Map3k1, Spata16, and Fgf5) were enriched in the MAPK signalling and Hedgehog signalling pathways, both of which are closely associated with hair growth regulation. Among these genes, Fgf5 encodes the FGF5 protein, which is a well-established regulator of hair growth. There was a nonsynonymous nucleotide substitution (T19234C) in the Fgf5 gene. At this locus, the C allele was present in all of the tested Angora rabbits, while the T allele was dominant in New Zealand and Rex rabbits. We further confirmed that the C allele was conserved in Angora rabbits by screening an additional 135 rabbits. Moreover, the results of functional predictions and co-immunoprecipitation revealed that the T19234C mutation impaired the binding capacity of FGF5 to its receptor FGFR1. CONCLUSIONS: We discovered that the homozygous missense mutation T19234C within Fgf5 might contribute to the long-hair trait of Angora rabbits by reducing its receptor binding capacity. This finding will provide new insights into the genetic basis underlying the genetic improvement of Angora rabbits and benefit the improvement of rabbit breeding in the future.

Proteomics Analysis of Lipid Metabolism and Inflammatory Response in the Liver of Rabbits fed on a High Cholesterol Diet.

In this study, we aimed to analyze the proteomics of the liver in rabbits on a high cholesterol diet (HCD). We randomly divided New Zealand white rabbits into the normal diet group and the HCD group. We established the atherosclerosis model and measured plasma cholesterol and triglycerides. The model was successfully established using ultrasound examination and histopathological staining of the intima of aorta and liver of the two groups of rabbits. The differential proteins in the rabbit liver were analyzed using Tandem Mass Tags proteomic analysis technology. Finally, we used western blot to verify the reliability of proteomics. The results showed that compared with the control group, the serum lipid levels of rats in the HCD group was significantly increased, and the pathological sections showed the formation of atherosclerotic plaques in the aorta, inflammation, and adipose lesions in the liver. Proteomic analysis of the liver revealed 149 differences in HCD-expressed protein, which is mainly involved in inflammation and regulation of lipid and sugar metabolism. In addition, we verified differentially expressed liver proteins in the HCD group using western blot. We found that HCD caused lipid accumulation, abnormal glucose metabolism, and inflammatory response in the liver.

A novel α Globin Gene Cluster Duplication, αααα380 Heterozygous ß0-Thal Variant, Leading to a Blood Transfusion-Dependent Phenotype.

To assess the effectiveness of three-level prevention and control of thalassemia, we routinely collect samples from transfusion-dependent individuals and perform genetic analysis. Here, we report on a 10-year-old boy requiring blood transfusions with routine thalassemia gene test results of αα/αα, and ßCD41/42/ßN, but he had thalassemia-like changes in his appearance and a high need for frequent blood transfusions, suggesting a case of thalassemia major in childhood. Given these equivocal results, samples from the family members were collected for further analysis. A multiplex ligation-dependent probe amplification assay was used to detect a multicopy number variant of the α globin gene cluster in the proband. The variant was detected as a long fragment repeat of 380 Kb using CNV assay technique, which contains the entire α globin gene cluster, describing it as αααα380/αα. Analysis of family members suggested that both the brother and mother of the proband carried the variant, and both MCV and MCH values were reduced in carriers. Individuals carrying multiple copy number variants of the α globin gene cluster exist in the population. Individuals carrying such variants who are also heterozygous for the ß0 thalassemia variant result in an imbalance in the α/ß chain ratio, potentially leading to the creation of individuals with a severe anemia genotype. Most secondary prevention and control laboratories currently do not include variants with increased α gene copy number in their testing, which is one of the blind spots of prevention and control efforts. In order to provide more accurate genetic counseling to test subjects, especially in regions with high rates of thalassemia carriage, testing laboratories should pay attention to individual genotype-phenotype matches to avoid the under-detection of such variants.

Trends and characteristics of COVID-19 and cardiovascular disease related studies.

Introduction: The new coronavirus has caused a pandemic that has infected hundreds of millions of people around the world since its outbreak. But the cardiovascular damage caused by the new coronavirus is unknown. We have analyzed the current global scenario and the general pattern of growth. After summarizing the known relationship between cardiovascular diseases and new coronary pneumonia, relevant articles are analyzed through bibliometrics and visualization. Methods: Following our pre-designed search strategy, we selected publications on COVID-19 and cardiovascular disease in the Web of Science database. In our relevant bibliometric visualization analysis, a total of 7,028 related articles in the WOS core database up to 20th October 2022 were summarized, and the most prolific authors, the most prolific countries, and the journals and institutions that published the most articles were summarized and quantitatively analyzed. Results: SARS-CoV-2 is more infectious than SARS-CoV-1 and has significant involvement in the cardiovascular system in addition to pulmonary manifestations, with a difference of 10.16% (20.26%/10.10%) in the incidence of cardiovascular diseases. The number of cases increases in winter and decreases slightly in summer with temperature changes, but the increase in cases tends to break out of seasonality across the region as mutant strains emerge. The co-occurrence analysis found that with the progress of the epidemic, the research keywords gradually shifted from ACE2 and inflammation to the treatment of myocarditis and complications, indicating that the research on the new crown epidemic has entered the stage of prevention and treatment of complications. Conclusion: When combined with the current global pandemic trend, how to improve prognosis and reduce human body damage could become a research focus. At the same time, timely detection, prevention, and discovery of new mutant strains have also become key tasks in the fight against the epidemic, and full preparations have been made to prevent the spread of the next wave of mutant strains, and still need to continue to pay attention to the differential performance of the variant "omicron."

Regulation of endothelial-to-mesenchymal transition by histone deacetylase 3 posttranslational modifications in neointimal hyperplasia.

Background: Endothelial-to-mesenchymal transition (EndMT) is the process by which endothelial cells lose their specific markers and acquire mesenchymal or myofibroblastic phenotypes. Studies have demonstrated the importance of endothelial-derived vascular smooth muscle cells (VSMCs) through EndMT in neointimal hyperplasia. Histone deacetylases (HDACs) are epigenetic modification enzymes involved in the epigenetic control of important cellular functions. Recent studies found that HDAC3, a class I HDAC, causes posttranslational modifications, including deacetylation and decrotonylation. However, the effect of HDAC3 on EndMT in neointimal hyperplasia via posttranslational modifications remains to be seen. Therefore, we investigated the effects of HDAC3 on EndMT in carotid artery-ligated mice and human umbilical vein endothelial cells (HUVECs) and the underlying posttranslational modifications. Methods: HUVECs were treated with transforming growth factor (TGF)-ß1 or the inflammatory cytokine tumor necrosis factor (TNF)-α at different concentrations and durations. In HUVECs, HDAC3 expression, the expression of endothelial and mesenchymal markers, and posttranslational modifications were analyzed with Western blotting, quantitative real-time polymerase chain reaction (PCR), and immunofluorescence. C57BL/6 mice underwent left carotid artery ligation. Mice were treated with the HDAC3-selective inhibitor RGFP966 (10 mg/kg, i.p.) from 1 day before to 14 days after ligation. Then, the sections of the carotid arteries were examined histologically using hematoxylin and eosin (HE) and immunofluorescence staining. The carotid arteries from other mice were examined for the expression of EndMT markers and inflammatory cytokines. Furthermore, the acetylation and crotonylation of carotid arteries were immunostained in mice. Results: In HUVECs, TGF-ß1 and TNF-α induced EndMT by decreasing CD31 expression and increasing α-smooth muscle actin expression. TGF-ß1 and TNF-α also upregulated HDAC3 expression in HUVECs. The in vivo study in mice indicated that RGFP966 significantly alleviated neointimal hyperplasia of the carotid artery compared with vehicle treatment. Furthermore, RGFP966 suppressed EndMT and the inflammatory response in carotid artery-ligated mice. Further investigation revealed that HDAC3 regulated EndMT by posttranslational modifications of deacetylation and decrotonylation. Conclusions: These results suggest that HDAC3 regulates EndMT in neointimal hyperplasia through posttranslational modifications.

Genetic deficiency of protein inhibitor of activated STAT3 suppresses experimental abdominal aortic aneurysms.

Aim: Signal transducer and activator of transcription (STAT) signaling is critical for the pathogenesis of abdominal aortic aneurysms (AAAs). Though protein inhibitor of activated STAT3 (PIAS3) negatively modulates STAT3 activity, but its role in AAA disease remains undefined. Method: AAAs were induced in PIAS3 deficient (PIAS3-/-) and wild type (PIAS3+/+) male mice via transient intra-aortic elastase infusion. AAAs were assessed by in situ measurements of infrarenal aortic external diameters prior to (day 0) and 14 days after elastase infusion. Characteristic aneurysmal pathologies were evaluated by histopathology. Results: Fourteen days following elastase infusion, aneurysmal aortic diameter was reduced by an approximately 50% in PIAS3-/- as compared to PIAS3+/+ mice. On histological analyses, PIAS3-/- mice showed less medial elastin degradation (media score: 2.5) and smooth muscle cell loss (media score: 3.0) than those in PIAS3+/+ mice (media score: 4 for both elastin and SMC destruction). Aortic wall leukocyte accumulation including macrophages, CD4+ T cells, CD8+ T cells and B cells as well as mural neovessel formation were significantly reduced in PIAS3-/- as compared to PIAS3+/+ mice. Additionally, PIAS3 deficiency also downregulated the expression levels of matrix metalloproteinases 2 and 9 by 61% and 70%, respectively, in aneurysmal lesion. Conclusion: PIAS3 deficiency ameliorated experimental AAAs in conjunction with reduced medial elastin degradation and smooth muscle cell depletion, mural leukocyte accumulation and angiogenesis.

DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates.

Nonalcoholic steatohepatitis (NASH) prevalence is rising with no pharmacotherapy approved. A major hurdle in NASH drug development is the poor translatability of preclinical studies to safe/effective clinical outcomes, and recent failures highlight a need to identify new targetable pathways. Dysregulated glycine metabolism has emerged as a causative factor and therapeutic target in NASH. Here, we report that the tripeptide DT-109 (Gly-Gly-Leu) dose-dependently attenuates steatohepatitis and fibrosis in mice. To enhance the probability of successful translation, we developed a nonhuman primate model that histologically and transcriptionally mimics human NASH. Applying a multiomics approach combining transcriptomics, proteomics, metabolomics, and metagenomics, we found that DT-109 reverses hepatic steatosis and prevents fibrosis progression in nonhuman primates, not only by stimulating fatty acid degradation and glutathione formation, as found in mice, but also by modulating microbial bile acid metabolism. Our studies describe a highly translatable NASH model and highlight the need for clinical evaluation of DT-109.

Somatic Cell Nuclear Transfer in Rabbits.

Somatic cell nuclear transfer (SCNT) is a technology that enables differentiated somatic cells to acquire a totipotent state, thus making it of great value in developmental biology, biomedical research, and agricultural applications. Rabbit cloning associated with transgenesis has the potential to improve the applicability of this species for disease modeling, drug testing, and production of human recombinant proteins. In this chapter, we introduce our SCNT protocol for the production of live cloned rabbits.

Effect of Dietetic Obesity on Testicular Transcriptome in Cynomolgus Monkeys.

Obesity is a metabolic disorder resulting from behavioral, environmental and heritable causes, and can have a negative impact on male reproduction. There have been few experiments in mice, rats, and rabbits on the effects of obesity on reproduction, which has inhibited the development of better treatments for male subfertility caused by obesity. Nonhuman primates are most similar to human beings in anatomy, physiology, metabolism, and biochemistry and are appropriate subjects for obesity studies. In this investigation, we conducted a transcriptome analysis of the testes of cynomolgus monkeys on high-fat, high-fructose, and cholesterol-rich diets to determine the effect of obesity on gene expression in testes. The results showed that the testes of obese monkeys had abnormal morphology, and their testes transcriptome was significantly different from that of non-obese animals. We identified 507 differentially abundant genes (adjusted p value < 0.01, log2 [FC] > 2) including 163 up-regulated and 344 down-regulated genes. Among the differentially abundant genes were ten regulatory genes, including IRF1, IRF6, HERC5, HERC6, IFIH1, IFIT2, IFIT5, IFI35, RSAD2, and UBQLNL. Gene ontology (GO) and KEGG pathway analysis was conducted, and we found that processes and pathways associated with the blood testes barrier (BTB), immunity, inflammation, and DNA methylation in gametes were preferentially enriched. We also found abnormal expression of genes related to infertility (TDRD5, CLCN2, MORC1, RFX8, SOHLH1, IL2RB, MCIDAS, ZPBP, NFIA, PTPN11, TSC22D3, MAPK6, PLCB1, DCUN1D1, LPIN1, and GATM) and down-regulation of testosterone in monkeys with dietetic obesity. This work not only provides an important reference for research and treatment on male infertility caused by obesity, but also valuable insights into the effects of diet on gene expression in testes.

Urotensin II Enhances Advanced Aortic Atherosclerosis Formation and Delays Plaque Regression in Hyperlipidemic Rabbits.

Accumulated evidence shows that elevated urotensin II (UII) levels are associated with cardiovascular diseases. However, the role of UII in the initiation, progression, and regression of atherosclerosis remains to be verified. Different stages of atherosclerosis were induced in rabbits by a 0.3% high cholesterol diet (HCD) feeding, and either UII (5.4 µg/kg/h) or saline was chronically infused via osmotic mini-pumps. UII promoted atherosclerotic fatty streak formation in ovariectomized female rabbits (34% increase in gross lesion and 93% increase in microscopic lesion), and in male rabbits (39% increase in gross lesion). UII infusion significantly increased the plaque size of the carotid and subclavian arteries (69% increase over the control). In addition, UII infusion significantly enhanced the development of coronary lesions by increasing plaque size and lumen stenosis. Histopathological analysis revealed that aortic lesions in the UII group were characterized by increasing lesional macrophages, lipid deposition, and intra-plaque neovessel formation. UII infusion also significantly delayed the regression of atherosclerosis in rabbits by increasing the intra-plaque macrophage ratio. Furthermore, UII treatment led to a significant increase in NOX2 and HIF-1α/VEGF-A expression accompanied by increased reactive oxygen species levels in cultured macrophages. Tubule formation assays showed that UII exerted a pro-angiogenic effect in cultured endothelial cell lines and this effect was partly inhibited by urantide, a UII receptor antagonist. These findings suggest that UII can accelerate aortic and coronary plaque formation and enhance aortic plaque vulnerability, but delay the regression of atherosclerosis. The role of UII on angiogenesis in the lesion may be involved in complex plaque development.