Deconvolution of synovial myeloid cell subsets across pathotypes and role of COL3A1+ macrophages in rheumatoid arthritis remission.

Background: Monocyte/macrophage (Mo/Mp) is a critical cell population involved in immune modulation of rheumatoid synovitis (RA) across different pathotypes. This study aims to investigate the contribution of Mo/Mp clusters to RA activity, and the biological function of particular subtypes in RA remission. Methods: We integrated single-cell RNA sequencing datasets from 4 published and 1 in-house studies using Liger selected by comparison. We estimated the abundance of Mo/Mp subtypes in bulk RNA-seq data from the 81 patients of the Pathobiology of Early Arthritis Cohort (PEAC) using deconvolution analysis. Correlations between Mo/Mp subtypes and RA clinical metrics were assessed. A particular cell type was identified using multicolor immunofluorescence and flow cytometry in vivo and successfully induced from a cell line in vitro. Potential immune modulation function of it was performed using immunohistochemical staining, adhesion assay, and RT-qPCR. Results: We identified 8 Mo/Mp clusters. As a particular subtype among them, COL3A1+ Mp (CD68+, COL3A1+, ACTA2-) enriched in myeloid pathotype and negatively correlated with RA severity metrics in all pathotypes. Flow cytometry and multicolor immunofluorescence evidenced the enrichment and M2-like phenotype of COL3A1+ Mp in the myeloid pathotype. Further assays suggested that COL3A1+ Mp potentially attenuates RA severity via expressing anti-inflammatory cytokines, enhancing Mp adhesion, and forming a physical barrier at the synovial lining. Conclusion: This study reported unexplored associations between different pathologies and myeloid cell subtypes. We also identified a fibroblast-and-M2-like cluster named COL3A1+ Mp, which potentially contributes to synovial immune homeostasis. Targeting the development of COL3A1+ Mp may hold promise for inducing RA remission.

WNT4 promotes macrophage polarization via granulosa cell M-CSF and reduces granulosa cell apoptosis in endometriosis.

BACKGROUND: WNT4 gene polymorphism are common in endometriosis and may functionally link estrogen and estrogen receptor signaling. Previous study confirmed estrogen and estrogen receptor signaling recruit macrophage to promote the pathogenesis of endometriosis. To investigate the effect of WNT4 in endometriosis involved in macrophage polarization and whether WNT4 could reduce the apoptosis of granulosa cells. METHODS: An observational study consisting of 8 cases of women with endometriosis (diagnosed by surgery and histology) and 22 mice of endometriosis animal model was conducted. Granulosa cells were isolated from 16 patients with endometriosis and co-cultured with macrophage under WNT4 treatment using TUNEL assay, quantitative reverse transcription PCR, flow cytometry and ELISA analysis. 22 mice of endometriosis animal model confirmed the WNT4 treatment effects using histology and immunohistochemistry, Western blot and flow cytometry. RESULTS: We observed that the apoptotic proportion of granulosa cells was significantly decreased and M2 macrophage was significantly increased after WNT4 treatment during the granulosa cell and macrophage co-culture system. To reveal the underlying mechanism for this, we conducted a series of experiments and found that high expression of granulosa cell M-CSF led to the M2 polarization of macrophages. The animal model also suggested that the anti-apoptotic effect of WNT4 on granulosa cells were conducted by the M2 polarized macrophage. CONCLUSIONS: WNT4 could reduce granulosa cell apoptosis and improve ovarian reserve by promoting macrophage polarization in endometriosis. M-CSF secreted by granulosa cell after WNT4 treatment was the main mediator of macrophage polarization.

Ultrasound-based radiomics score for pre-biopsy prediction of prostate cancer to reduce unnecessary biopsies.

BACKGROUND: Patients undergoing prostate biopsies (PBs) suffer from low positive rates and potential risk for complications. This study aimed to develop and validate an ultrasound (US)-based radiomics score for pre-biopsy prediction of prostate cancer (PCa) and subsequently reduce unnecessary PBs. METHODS: Between December 2015 and March 2018, 196 patients undergoing initial transrectal ultrasound (TRUS)-guided PBs were retrospectively enrolled and randomly assigned to the training or validation cohort at a ratio of 7:3. A total of 1044 radiomics features were extracted from grayscale US images of each prostate nodule. After feature selection through the least absolute shrinkage and selection operator (LASSO) regression model, the radiomics score was developed from the training cohort. The prediction nomograms were developed using multivariate logistic regression analysis based on the radiomics score and clinical risk factors. The performance of the nomograms was assessed and compared in terms of discrimination, calibration, and clinical usefulness. RESULTS: The radiomics score consisted of five selected features. Multivariate logistic regression analysis demonstrated that the radiomics score, age, total prostate-specific antigen (tPSA), and prostate volume were independent factors for prediction of PCa (all p < 0.05). The integrated nomogram incorporating the radiomics score and three clinical risk factors reached an area under the curve (AUC) of 0.835 (95% confidence interval [CI], 0.729-0.941), thereby outperforming the clinical nomogram which based on only clinical factors and yielded an AUC of 0.752 (95% CI, 0.618-0.886) (p = 0.04). Both nomograms showed good calibration. Decision curve analysis indicated that using the integrated nomogram would add more benefit than using the clinical nomogram. CONCLUSION: The radiomics score was an independent factor for pre-biopsy prediction of PCa. Addition of the radiomics score to the clinical nomogram shows incremental prognostic value and may help clinicians make precise decisions to reduce unnecessary PBs.

Bacterial Vaginosis Decreases the Risk of Cervical Cytological Abnormalities.

Genital tract infections, including vulvovaginal candidiasis and bacterial vaginosis, have emerged as potential modulators of persistent human papillomavirus (HPV) infections causing cervical cytologic abnormalities and cervical cancer. This study aimed to investigate whether vulvovaginal candidiasis or bacterial vaginosis had an additional effect on HPV infection and thus caused such abnormalities. ThinPrep cytologic tests were used to detect cytologic abnormalities, vulvovaginal candidiasis, and bacterial vaginosis in 14,679 women. Cytologic abnormalities included atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, atypical squamous cells-cannot exclude HSIL, and squamous cell carcinoma. Logistic regression Model 1 (univariate regression) and Model 2 (multivariate logistic regression analysis adjusted for age combined with HPV infection) were used to analyze the association between bacterial vaginosis and cytologic abnormalities, or vulvovaginal candidiasis and cytologic abnormalities, alone or in the presence of HPV infection. Bacterial vaginosis infection rates were found to be significantly higher in the cytology-negative group among all participants and those with HPV infection (P = 0.003, P < 0.001, respectively). Analyses using Model 1 and Model 2 both pointed to bacterial vaginosis as a protective factor against cytologic abnormalities for all participants (OR = 0.36, 0.17, respectively, P < 0.05) and for HPV-infected participants (OR = 0.17, 0.16, respectively, P < 0.05). Neither vulvovaginal candidiasis nor vulvovaginal candidiasis + HPV was significantly associated with the incidence of cytologic abnormalities based on Model 1 (OR = 0.94, 0.71, respectively, P > 0.05) and Model 2 (OR = 0.91, 0.74, respectively, P > 0.05). Furthermore, neither vulvovaginal candidiasis nor bacterial vaginosis increased the incidence of cytologic abnormalities regardless of HPV infection status, while bacterial vaginosis might possibly prevent cytologic abnormalities in women coinfected by HPV. PREVENTION RELEVANCE: Neither vulvovaginal candidiasis nor bacterial vaginosis was found to increase the incidence of cervical cytologic abnormalities with or without the presence of HPV. On the contrary, bacterial vaginosis may play a role in preventing cytologic abnormalities in women with HPV coinfection.

Tumor-associated macrophages induced spheroid formation by CCL18-ZEB1-M-CSF feedback loop to promote transcoelomic metastasis of ovarian cancer.

BACKGROUND: Ovarian cancer (OvCa)-tumor-associated macrophages (TAMs) spheroids are abundantly present within ascites of high malignant patients. This study investigated the mutual interaction of OvCa cells and TAMs in the spheroids. METHODS: Three-dimensional coculture system and transwell coculture system were created to mimic the OvCa and TAMs in spheroids and in disassociated state. Transwell-migration assay and scratch wound healing assay were used to measure the invasive and migratory capacity. Western blot, quantitative reverse transcription-PCR and immunostaining were used to measure the mesenchymal and epithelial markers. Flow cytometry was used to assess the polarization of TAMs. Also, the differential gene expression profile of OvCa cells and OvCa cells from spheroids were tested by RNA-sequence. Finally, the ovarian mice models were constructed by intraperitoneal injection of ID8 or OvCa-TAMs spheroids. RESULTS: Our results indicated that the formation of OvCa-TAMs spheroids was positive related to the malignancy of OvCa cells. M2-TAMs induced the epithelial-mesenchymal transition of OvCa cells by releasing chemokine (C-C motif) ligand 18 (CCL18) in the spheroids. While, CCL18 induced macrophage colony-stimulating factor (M-CSF) transcription in OvCa cells through zinc finger E-box-binding homeobox 1 (ZEB1). This study further indicated that M-CSF secreted by OvCa cells drived the polarization of M2-TAMs. Therefore, a CCL18-ZEB1-M-CSF interacting loop between OvCa cells and TAMs in the spheroids was identified. Moreover, with blocking the expression of ZEB1 in the OvCa cell, the formation of OvCa-TAMs spheroids was impeded. In the ovarian mice models, the formation of OvCa-TAMs spheroids in the ascites was promoted by overexpressing of ZEB1 in OvCa cells, which resulted in faster and earlier transcoelomic metastasis. CONCLUSION: These findings suggested that the formation of OvCa-TAMs spheroids resulted in aggressive phenotype of OvCa cells, as a specific feedback loop CCL18-ZEB1-M-CSF in it. Inhibition of ZEB1 reduced OvCa-TAMs spheroids in the ascites, impeding the transcoelomic metastasis and improving the outcome of ovarian patients.

GnRHa/Stanozolol Combined Therapy Maintains Normal Bone Growth in Central Precocious Puberty.

Background: Gonadotropin-releasing hormone agonist (GnRHa) is the gold standard in the treatment of Central Precocious Puberty (CPP) with progressive puberty and accelerative growth. However, GnRHa treatment is reported to result in growth deceleration and prevents growth plate development which leads to a reduction in height velocity. Stanozolol (ST) has been used to stimulate growth in patients with delayed growth and puberty, nevertheless, the effects and mechanisms of ST on CPP with GnRHa treatment are currently unclear. Methods and Results: In the current study, we recorded the following vital observations that provided insights into ST induced chondrogenic differentiation and the maintenance of normal growth plate development: (1) ST efficiently prevented growth deceleration and maintained normal growth plate development in rats undergoing GnRHa treatment; (2) ST suppressed the inhibitory effect of GnRHa to promote chondrogenic differentiation; (3) ST induced chondrogenic differentiation through the activation of the JNK/c-Jun/Sox9 signaling pathway; (4) ST promoted chondrogenic differentiation and growth plate development through the JNK/Sox9 signaling pathway in vivo. Conclusions: ST mitigated the inhibitory effects of GnRHa and promoted growth plate development in rats. ST induced the differentiation of chondrocytes and maintained normal growth plate development through the activation of JNK/c-Jun/Sox9 signaling. These novel findings indicated that ST could be a potential agent for maintaining normal bone growth in cases of CPP undergoing GnRHa treatment.

Low basal serum testosterone level is detrimental to the embryo implantation in the patients with severe endometriosis.

AIM: Androgens have been reported to be associated with female fertility. The mean serum testosterone concentration in the patients with endometriosis was reported to be significantly lower than that without endometriosis. Our study was designed to investigate the influence of basal serum testosterone levels on the clinical outcome of in vitro fertilization (IVF) in the patients with III-IV stage endometriosis. METHODS: This retrospective cohort study included 407 patients with III-IV stage endometriosis diagnosed by laparoscopic surgery. We studied the association of the basal serum testosterone level and the reproductive outcome of IVF. RESULTS: The basal serum testosterone concentration was significantly higher in the pregnant group of patients with III-IV stage endometriosis. The further analyses demonstrated that the implantation rate of the basal serum testosterone concentration < 0.305 ng/mL group was significantly lower than the testosterone ≥ 0.305 ng/mL group (24.1% vs. 32.7%, p = 0.007). The clinical pregnancy and live birth rate of the basal serum testosterone < 0.305 ng/mL group were also lower than that of the testosterone ≥ 0.305 ng/mL group. Both initial and total dose of gonadotropins in the testosterone <0.305 ng/mL group are significantly higher than that of the testosterone ≥0.305 ng/mL group. CONCLUSIONS: Our study demonstrated, for the first time, that the basal serum testosterone <0.305 ng/mL had an adverse impact on pregnancy outcomes of IVF-embryo transfer in the patients with III-IV stage endometriosis. Besides, the basal serum testosterone is also helpful in making individual stimulation protocol for the patients with advanced endometriosis before entering IVF cycles.

Protective Effects of Reduced Glutathione and Ulinastatin on Xeno-transplanted Human Ovarian Tissue Against Ischemia and Reperfusion Injury.

Recently, transplantation of cryopreserved ovarian tissue is the method for fertility preservation for oncologic and nononcologic reasons. The main challenge of ovarian cryopreservation followed by transplantation is that ischemia reperfusion injury (IRI) induced the loss of follicles. The aim of this study was to evaluate the effects of glutathione (GSH), ulinastatin (UTI) or both (GSH+UTI) on preventing ischemia reperfusion-induced follicles depletion in ovarian grafts.Ovarian fragments were collected from 20 women aged 29±6 years. Frozen-thawed human ovarian tissue was xenografted into SCID mice, at the same time GSH, UTI and GSH+UTI was administrated respectively. The ovarian grafts were collected at the 1st, 3rd, 7th, 14th, 28th, 56th, and 85th day after xenotransplantation. Follicle survival rate was measured by H&E staining and Live/Dead staining. Angiogenic activity and macrophage recruitment was evidenced by immunohistochemical staining. The oxidative stress and inflammatory cytokines in human ovarian xenografts were measured by real-time PCR. The results indicated that after the treatments of GSH, UTI and GSH+UTI in the hosts, follicular survival in ovarian grafts were improved. The level of VEGF, CD31, and antioxidant enzymes superoxide dismutase 1 and superoxide dismutase 2 in ovarian grafts were increased. Accumulation of macrophages, level of IL6 and TNF-α, as well as malondialdehyde was decreased in ovarian grafts from treated groups. In conclusion, administration of GSH, UTI and GSH+UTI decreased the depletion of follicles in human grafts post-transplantation by inhibiting IRI-induced antiangiogenesis, oxidative stress and inflammation.

Severe cervical inflammation and high-grade squamous intraepithelial lesions: a cross-sectional study.

PURPOSE: Inflammation has been reported as a facilitator in cervical oncogenesis, but the correlation between inflammation and cytological abnormality remains uncertain. The aim of this study was to investigate the correlation between inflammation and cytological abnormality. METHODS: ThinPrep cytological test (TCT) was used to detect cervical cytological abnormalities and inflammation degrees of 46,255 women in this prospective cross-sectional study. Histopathological examination was used to define the cervical intraepithelial neoplasia (CIN) in patients with cervical cytological abnormalities. RESULTS: The study revealed that 8.87% (4102/46,255) of TCT results had cytological abnormalities. The 4102 included cases were classified as the case group, including atypical squamous cells (ASC), low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). Women with negative intraepithelial lesion or malignancy (NILM) were classified as the control group. About 88.83% (3644/4102) of women with cytological abnormalities showed inflammations. The rate of severe inflammation was significantly higher in the case group than the control group (23.86% vs. 2.0%, P = 0.000). Our results also showed that patients with severe inflammation had a significantly increasing incidence of cytological abnormality by 12.598 times and elevated the risk of HSIL by 756.47 times, compared to the inflammation negative group. CONCLUSION: Severe inflammation was positively related to HSIL. Patients with severe cervical inflammation should be given more follow-ups and regular examinations and treated more carefully than those with mild or no inflammation.

Transplantation of Wnt5a-modified NSCs promotes tissue repair and locomotor functional recovery after spinal cord injury.

Traditional therapeutic strategies for spinal cord injury (SCI) are insufficient to repair locomotor function because of the failure of axonal reconnection and neuronal regeneration in the injured central nervous system (CNS). Neural stem cell (NSC) transplantation has been considered a potential strategy and is generally feasible for repairing the neural circuit after SCI; however, the most formidable problem is that the neuronal differentiation rate of NSCs is quite limited. Therefore, it is essential to induce the neuronal differentiation of NSCs and improve the differentiation rate of NSCs in spinal cord repair. Our results demonstrate that both Wnt5a and miRNA200b-3p could promote NSC differentiation into neurons and that Wnt5a upregulated miRNA200b-3p expression through MAPK/JNK signaling to promote NSC differentiation into neurons. Wnt5a could reduce RhoA expression by upregulating miRNA200b-3p expression to inhibit activation of the RhoA/Rock signaling pathway, which has been reported to suppress neuronal differentiation. Overexpression of RhoA abolished the neurogenic capacity of Wnt5a and miRNA200b-3p. In vivo, miRNA200b-3p was critical for Wnt5a-induced NSC differentiation into neurons to promote motor functional and histological recovery after SCI by suppressing RhoA/Rock signaling. These findings provide more insight into SCI and help with the identification of novel treatment strategies.

Wnt4-modified NSC transplantation promotes functional recovery after spinal cord injury.

Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction, yet there are no effective therapies currently due to the failure of reconstructing the interruption of the neuroanatomical circuit. While neural stem cell (NSC) transplantation has been considered a potential strategy to repair the neural circuit after SCI, the efficacy of this strategy remains unproven. The main reason is that most of the transplanted NSC differentiates into astrocyte rather than neuron in the microenvironment of SCI. Our results demonstrated that Wnt4 significantly promotes the differentiation of NSC into neuron by activating both ß-catenin and MAPK/JNK pathways and suppressing the activation of Notch signaling, which is acknowledged as prevention of NSC differentiation into neuron, through downregulating NICD expression, translocating and preventing the combination of NICD and RbpJ in nucleus. In addition, Wnt4 rescues the negative effect of Jagged, the ligand of Notch signaling, to promote neuronal differentiation. Moreover, in vivo study, transplantation of Wnt4-modified NSC efficaciously repairs the injured spinal cord and recovers the motor function of hind limbs after SCI. This study sheds new light into mechanisms that Wnt4-modified NSC transplantation is sufficient to repair the injured spinal cord and recover the motor dysfunction after SCI.

Investigation Of Vitamin B12-Modified Amphiphilic Sodium Alginate Derivatives For Enhancing The Oral Delivery Efficacy Of Peptide Drugs.

PURPOSE: Peptide drugs have been used in therapy various diseases. However, the poor bioavailability of peptide drugs for oral administration has limited their clinical applications, on account of the acidic environment and digestive enzymes inside the human gastrointestinal tract. To enhance stability in the human gastrointestinal tract, bioavailability, and targeted drug delivery of peptide drugs through oral administration, a vitamin B12-modified amphiphilic sodium alginate derivative (CSAD-VB12) was synthesized. MATERIALS AND METHODS: A vitamin B12-modified amphiphilic sodium alginate derivative (CSAD-VB12) was synthesized via the N,N'-dicyclohexylcarbodiimide active method at room temperature, and then characterized using FTIR and 1H NMR spectroscopy. Insulin was used as a model peptide drug and the insulin-loaded CSAD-VB12 (CSAD-VB12/insulin) nanoparticles with negative zeta potentials were prepared in PBS (pH=7.4). Scanning electron microscopy was used to observe CSAD-VB12/insulin as spherical nanoparticles. The CSAD-VB12 derivatives and CSAD-VB12/insulin nanoparticles displayed nontoxicity towards the human colon adenocarcinoma (Caco-2) cells by CCK-8 test. Caco-2 cell model was used to measure the apparent permeability (Papp) of insulin, CSAD/insulin and CSAD-VB12/insulin. Furthermore, confocal was used to confirm the endocytosis of intestinal enterocytes. Type 1 diabetes mice were used to evaluate the intestinal absorption and retention effect of test nanoparticles. RESULTS: They were observed as spherical nanoparticles in the size of 30-50 nm. The CSAD-VB12 derivatives and CSAD-VB12/insulin nanoparticles displayed nontoxicity towards the human colon adenocarcinoma (Caco-2) cells. Comparing with insulin and the CSAD/insulin nanoparticles, the CSAD-VB12/insulin nanoparticles exhibited higher permeation ability through intestinal enterocytes in the Caco-2 cell model. Oral administration of the CSAD-VB12/insulin nanoparticles to Type 1 diabetic mice yields higher intestinal retention effect, targeted absorption, and outstanding efficacy. CONCLUSION: CSAD-VB12 derivatives enhance the small intestinal absorption efficacy and retention of peptide by oral administration, which indicated that it could be a promising candidate for oral peptide delivery in the prospective clinical application.

Inhibition of Notch1 signaling promotes neuronal differentiation and improves functional recovery in spinal cord injury through suppressing the activation of Ras homolog family member A.

Neural stem cells (NSCs) transplantation represents a promising strategy for the repair of injured neurons, since NSCs not only produce multiple neurotrophic growth factors but also differentiate into mature cells to replace damaged cells. Previous studies have shown that Notch signaling pathway had negative effects on neuronal differentiation; however, the precise mechanism remained inadequately understood. This research aimed to investigate whether inhibition of Notch1 signaling promotes neuronal differentiation and improves functional recovery in rat spinal cord injury through suppressing the activation of Ras homolog family member A (RhoA). QPCR, western blot, and immunofluorescence experiments were used to analyze Notch1 signaling pathways, RhoA, Ras homologous -associated coiled-coil containing protein kinase 1 (ROCK1), cleaved caspased-3, and neuronal/astrocytic differentiation markers. The expression of RhoA and ROCK1 was inhibited by lentivirus or specific biochemical inhibitors. In spinal cord injury (SCI), motor function was assessed by hind limbs movements and electrophysiology. Tissue repairing was measured by immunofluorescence, Nissl staining, Fluorogold, HE staining, QPCR, western blot, and magnetic resonance imaging (MRI) experiments. Our results demonstrate that inhibition of Notch1 in NSCs can promote the differentiation of NSCs to neurons. Knockdown of RhoA and inhibition of ROCK1 both can promote neuronal differentiation through inhibiting the activation of Notch1 signaling pathway in NSCs. In SCI, silencing RhoA enhanced neuronal differentiation and improved tissue repairing/functional recovery by inhibiting the activation of Notch1 signaling pathway. Since Notch1 inhibits neuronal differentiation through activating the RhoA/ROCK1 signaling pathway in NSCs, our data suggest that the Notch1/RhoA/ROCK1/Hes1/Hes5 signaling pathway may serve as a novel target for the treatment of SCI.

Ghrelin attenuates sepsis-induced acute lung injury by inhibiting the NF-κB, iNOS, and Akt signaling in alveolar macrophages.

Ghrelin has proven to be protective against sepsis-induced acute lung injury (ALI) via anti-inflammatory effects. However, its mechanisms remain poorly understood. Alveolar macrophages (AMs) play a key role in mediating inflammatory responses during sepsis-induced ALI by secretion of cytokines and chemokines. This study was undertaken to investigate whether ghrelin suppresses inflammatory effects of AMs and therefore may help to attenuate sepsis-induced ALI. A sepsis model in rats was achieved using cecal ligation and puncture. Ghrelin treatment markedly improved histopathological changes in the lungs and reduced pulmonary inflammation in septic rats. NF-κB translocation and p-Akt and inducible nitric oxide synthase (iNOS) activities in AMs from septic rats were suppressed by ghrelin. In vitro data indicated that ghrelin decreased the levels of LPS-induced IL-1ß, TNF-α, and IL-6, NF-κB translocation, and iNOS and Akt activities of AMs. Furthermore, the NF-κB/iNOS pathway or Akt signaling was positively correlated with LPS-induced inflammatory production of AMs in vitro. In conclusion, ghrelin exerts a protective role against sepsis-induced ALI probably by reducing the production of inflammatory cytokines from AMs via inhibition of the NF-κB/iNOS pathway or Akt signaling.

CD34+KLF4+ Stromal Stem Cells Contribute to Endometrial Regeneration and Repair.

The regenerative capacity of the human endometrium requires a population of local stem cells. However, the phenotypes, locations, and origin of these cells are still unknown. In a mouse menstruation model, uterine stromal SM22α+-derived CD34+KLF4+ stem cells are activated and integrate into the regeneration area, where they differentiate and incorporate into the endometrial epithelium; this process is correlated with enhanced protein SUMOylation in CD34+KLF4+ cells. Mice with a stromal SM22α-specific SENP1 deletion (SENP1smKO) exhibit accelerated endometrial repair in the regeneration model and develop spontaneous uterine hyperplasia. Mechanistic studies suggest that SENP1 deletion induces SUMOylation of ERα, which augments ERα transcriptional activity and proliferative signaling in SM22α+CD34+KLF4+ cells. These cells then transdifferentiate to the endometrial epithelium. Our study reveals that CD34+KLF4+ stromal-resident stem cells directly contribute to endometrial regeneration, which is regulated through SENP1-mediated ERα suppression.

Scutellarin Prevents Angiogenesis in Diabetic Retinopathy by Downregulating VEGF/ERK/FAK/Src Pathway Signaling.

BACKGROUND: Diabetic retinopathy (DR) is a serious microvascular complication of diabetes. This study demonstrates the antiangiogenic effects of scutellarin (SCU) on high glucose- and hypoxia-stimulated human retinal endothelial cells (HRECs) and on a diabetic rat model by oral administration. The antiangiogenic mechanisms of SCU in vitro and in vivo were investigated. METHOD: HRECs were cultured in high glucose- (30 mM D-glucose) and hypoxia (cobalt chloride-treated)-stimulated diabetic condition to evaluate the antiangiogenic effects of SCU by CCK-8 test, cell migration experiment (wound healing and transwell), and tube formation experiment. A streptozotocin-induced type II diabetic rat model was established to measure the effects of oral administration of SCU on protecting retinal microvascular dysfunction by Doppler waveforms and HE staining. We further used western blot, luciferase reporter assay, and immunofluorescence staining to study the antiangiogenic mechanism of SCU. The protein levels of phospho-ERK, phospho-FAK, phospho-Src, VEGF, and PEDF were examined in HRECs and retina of diabetic rats. RESULT: Our results indicated that SCU attenuated diabetes-induced HREC proliferation, migration, and tube formation and decreased neovascularization and resistive index in the retina of diabetic rats by oral administration. SCU suppressed the crosstalk of phospho-ERK, phospho-FAK, phospho-Src, and VEGF in vivo and in vitro. CONCLUSIONS: These results suggested that SCU can be an oral drug to alleviate microvascular dysfunction of DR and exerts its antiangiogenic effects by inhibiting the expression of the crosstalk of VEGF, p-ERK, p-FAK, and p-Src.

17ß-estradiol upregulates striatin protein levels via Akt pathway in human umbilical vein endothelial cells.

17ß-estradiol (E2) has been shown to have beneficial effects on the cardiovascular system. We previously demonstrated that E2 increases striatin levels and inhibits migration in vascular smooth muscle cells. The objective of the present study was to investigate the effects of E2 on the regulation of striatin expression in human umbilical vein endothelial cells (HUVECs). We demonstrated that E2 increased striatin protein expression in a dose- and time-dependent manner in HUVECs. Pretreatment with ICI 182780 or the phosphatidylinositol-3 kinase inhibitor, wortmannin, abolished E2-mediated upregulation of striatin protein expression. Treatment with E2 resulted in Akt phosphorylation in a time-dependent manner. Moreover, silencing striatin significantly inhibited HUVEC migration, while striatin overexpression significantly promoted HUVEC migration. Finally, E2 enhanced HUVEC migration, which was inhibited by silencing striatin. In conclusion, our results demonstrated that E2-mediated upregulation of striatin promotes cell migration in HUVECs.

3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells.

Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated spheroid and clinical pathology of ovarian cancer, and that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in orthotopic mouse models. We have established an in vitro spheroid formation assay using a 3D co-culture system in which mouse GFP+F4/80+CD206+ TAMs isolated from spheroids of ovarian cancer-bearing donor tomatolysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. As transcoelomic metastasis is also associated with many other cancers such as pancreatic and colon cancers, TAM-mediated spheroid formation assay would provide a useful approach to define the molecular mechanism and therapeutic targets for ovarian cancer and other transcoelomic metastasis cancers.

Enhancement of scutellarin oral delivery efficacy by vitamin B12-modified amphiphilic chitosan derivatives to treat type II diabetes induced-retinopathy.

BACKGROUND: Diabetic retinopathy is the most common complication in diabetic patients relates to high expression of VEGF and microaneurysms. Scutellarin (Scu) turned out to be effective against diabetes related vascular endothelial cell dysfunction. However, its clinical applications have been limited by its low bioavailability. In this study, we formulated and characterized a novel intestinal target nanoparticle carrier based on amphiphilic chitosan derivatives (Chit-DC-VB12) loaded with scutellarin to enhance its bioavailability and then evaluated its therapeutic effect in experimental diabetic retinopathy model. RESULTS: Chit-DC-VB12 nanoparticles showed low toxicity toward the human colon adenocarcinoma (Caco-2) cells and zebra fish within concentration of 250 µg/ml, owing to good biocompatibility of chitosan. The scutellarin-loaded Chit-DC-VB12 nanoparticles (Chit-DC-VB12-Scu) were then prepared by self-assembly in aqueous solution. Scanning electron microscopy and dynamic light scattering analysis indicated that the Chit-DC-VB12-Scu nanoparticles were spherical particles in the sizes ranging from 150 to 250 nm. The Chit-DC-VB12-Scu nanoparticles exhibited high permeation in Caco-2 cell, indicated it could be beneficial to be absorbed in humans. We also found that Chit-DC-VB12 nanoparticles had a high cellular uptake. Bioavailability studies were performed in Sprague-Dawley rats, which present the area under the curve of scutellarin of Chit-DC-VB12-Scu was two to threefolds greater than that of free scutellarin alone. Further to assess the therapeutic efficacy of diabetic retinopathy, we showed Chit-DC-VB12-Scu down-regulated central retinal artery resistivity index and the expression of angiogenesis proteins (VEGF, VEGFR2, and vWF) of retinas in type II diabetic rats. CONCLUSIONS: Chit-DC-VB12 nanoparticles loaded with scutellarin have better bioavailability and cellular uptake efficiency than Scu, while Chit-DC-VB12-Scu nanoparticles alleviated the structural disorder of intraretinal neovessels in the retina induced by diabetes, and it also inhibited the retinal neovascularization via down-regulated the expression of angiogenesis proteins. In conclusion, the Chit-DC-VB12 nanoparticles enhanced scutellarin oral delivery efficacy and exhibited potential as small intestinal target promising nano-carriers for treatment of type II diabetes induced-retinopathy.

Curcumin Ameliorates Reserpine-Induced Gastrointestinal Mucosal Lesions Through Inhibiting IκB-α/NF-κB Pathway and Regulating Expression of Vasoactive Intestinal Peptide and Gastrin in Rats.

The objective of our study was to investigate whether curcumin protects against reserpine-induced gastrointestinal mucosal lesions (GMLs) in rats and to explore the mechanism of curcumin's action. Sprague-Dawley rats were randomly divided into four groups: control group, reserpine-treated group, reserpine treatment group with curcumin at high dose (200 mg/kg), and reserpine treatment group with curcumin at low dose (100 mg/kg). Rats in reserpine-treated group were induced by intraperitoneally administered reserpine (0.5 mg/kg) for 28 days. TUNEL staining and hematoxylin and eosin staining were used to evaluate the apoptotic cells and morphologic changes. In addition, to explore the mechanism of curcumin in protecting GMLs, we used serum of experimental rats to assess the level of vasoactive intestinal peptide (VIP), gastrin, interleukin-6, interleukin-10, tumor necrosis factor-α and interferon-γ by ELISA and radioimmunoassay. The protein levels of NF-κB, p-IκB-α, IκB-α, Bcl-2, Bax, and cleaved-caspase-3 were examined by western blot analysis. Data were analyzed with SPSS 19.0 software package. Curcumin treatment prevented tissue damage and cell death in the reserpine-treated rats and effectively decreased inflammatory response and balanced the expression of VIP and gastrin in the reserpine-treated rats. NF-κB, p-IκB-α, Bax, and cleaved-caspase-3 were increased in the reserpine group, but the curcumin high-dose group inhibited them. Curcumin can target the IκB-α/NF-κB pathway to inhibit inflammatory response and regulate the level of VIP and gastrin in reserpine-induced GML rats.