[Progress in gene-modified stem cell therapy for penile erectile dysfunction].

Erectile dysfunction (ED) is a common disorder in men and lacks effective treatment. Stem cell (SC) possess the potential in self-renewal and multi-directional differentiation, and secrete a variety of active substances. Stem cell therapy, as a promising option for the treatment of ED, is a focus in regenerative medicine research. However, the effect of stem cell therapy alone on ED is limited due to the complexity of the condition and the limitations of SC. Gene modification can enhance the performance of SC in multiple aspects and play the role of targeted genes, and therefore can better improve ED and its related pathological changes than SC therapy alone. In recent years, gene-modified stem cell therapy has become a hotspot in andrological research. This review summarizes the advances in the studies of gene-modified SC in the treatment of ED, aiming to provide some ideas for further research.

The impact of donor hepatitis B virus infection on transplant outcomes in deceased donor kidney transplantation recipients.

Background: The use of hepatitis B virus (HBV)-positive donor kidneys to expand the donor pool has been implemented, but limited evidence exists regarding their impact on transplant outcomes. This study aimed to investigate the effects of donor HBV infection on transplant outcomes. Methods: Donor and recipient data between 2015 and 2021 were collected. A total of 743 kidney transplant cases were screened, including 94 donor hepatitis B surface antigen (HBsAg)+/recipient HBsAg- (D+R-) and 649 donor HBsAg-/recipient HBsAg- (D-R-) cases. The analysis endpoints included recipient HBV infection, delayed graft function (DGF), peak estimated glomerular filtration rate (eGFR) within 12 months, recipient survival, and death-censored graft survival (DCGS). Results: The D+R- group had a significantly higher risk of HBV infection compared to the D-R- group (6/72 vs. 3/231; relative risk, 6.4; p = 0.007). The risk of HBV transmission decreased significantly with increasing hepatitis B surface antibody (HBsAb) titer (p for trend = 0.003). Furthermore, the D+R- group did not exhibit an increased risk of DGF compared to the D-R- group (odds ratio, 0.70; p = 0.51) in the multivariable mixed model. Both groups had similar peak eGFR within 12 months (ß = 1.01, p = 0.71), and this had no impact on patient survival (hazard ratio [HR], 0.36; p = 0.10) and DCGS (HR, 0.79, p = 0.59) in the shared-frailty Cox model. Conclusion: The use of HBsAg-positive donor kidneys appears relatively safe for HBV-immunized recipients in the short term. D+R- does not negatively affect graft function recovery and provides comparable posttransplant outcomes. Maintaining an HBsAb titer over 100 IU/L before transplantation is critical to reduce the risk of HBV transmission.

Caveolin-1 scaffolding domain-derived peptide enhances erectile function by regulating oxidative stress, mitochondrial dysfunction, and apoptosis of corpus cavernosum smooth muscle cells in rats with cavernous nerve injury.

AIM: Increased corpus cavernosum smooth muscle cells (CCSMCs) apoptosis in the penis due to cavernous nerve injury (CNI) is a crucial contributor to erectile dysfunction (ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has been found to exert potential antiapoptotic properties. However, whether CSD peptide can alleviate CCSMCs apoptosis and ED in CNI rats remains unknown. The study aimed to determine whether CSD peptide can improve bilateral CNI-induced ED (BCNI-ED) by enhancing the antiapoptotic processes of CCSMCs. MAIN METHODS: Fifteen 10-week-old male Sprague-Dawley (SD) rats were randomly classified into three groups: sham surgery (Sham) group and BCNI groups that underwent saline or CSD peptide treatment respectively. At 3 weeks postoperatively, erectile function was assessed and the penis tissue was histologically examined. Furthermore, an in vitro model of CCSMCs apoptosis was established using transforming growth factor-beta 1 (TGF-ß1) to investigate the mechanism of CSD peptide in treating BCNI-ED. KEY FINDINGS: In BCNI rats, CSD peptide significantly prevented ED and decreased oxidative stress, the Bax/Bcl-2 ratio, and the levels of caspase3. TGF-ß1-treated CCSMCs exhibited severe oxidative stress, mitochondrial dysfunction, and apoptosis. However, CSD peptide partially reversed these alterations. SIGNIFICANCE: Exogenous CSD peptide could improve BCNI-ED by inhibiting oxidative stress, the Bax/Bcl-2 ratio, and caspase3 expression in penile tissue. The underlying mechanism might involve the regulatory effects of CSD peptide on oxidative stress, mitochondrial dysfunction, and apoptosis of CCSMCs following CNI. This study highlights CSD peptide as an effective therapy for post-radical prostatectomy ED (pRP-ED).

MicroRNA-145 Gene Modification Enhances the Retention of Bone Marrow-Derived Mesenchymal Stem Cells within Corpus Cavernosum by Targeting Krüppel-Like Factor 4.

PURPOSE: The poor retention and ambiguous differentiation of stem cells (SCs) within corpus cavernosum (CC) limit the cell application in erectile dysfunction (ED). Herein, the effects and mechanism of microRNA-145 (miR-145) gene modification on modulating the traits and fate of bone marrow-derived mesenchymal stem cells (BMSCs) were investigated. MATERIALS AND METHODS: The effects of miR-145 on cell apoptosis, proliferation, migration, and differentiation were determined by flow cytometry, cell counting kit-8, transwell assays and myogenic induction. Then, the age-related ED rats were recruited to four groups including phosphate buffer saline, BMSC, vector-BMSC, overexpressed-miR-145-BMSC groups. After cell transplantation, the CC were harvested and prepared to demonstrate the retention and differentiation of BMSCs by immunofluorescent staining. Then, the target of miR-145 was verified by quantitative real-time polymerase chain reaction and immunohistochemical. After that, APTO-253, as an inducer of Krüppel-like factor 4 (KLF4), was introduced for rescue experiments in corpus cavernosum smooth muscle cells (CCSMCs) under the co-culture system. RESULTS: In vitro, miR-145 inhibited the migration and apoptosis of BMSCs and promoted the differentiation of BMSCs into smooth muscle-like cells with stronger contractility. In vivo, the amount of 5-ethynyl-2'-deoxyuridine (EdU)+cells within CC was significantly enhanced and maintained in the miR-145 gene modified BMSC group. The EdU/CD31 co-staning was detected, however, no co-staining of EdU/α-actin was observed. Furthermore, miR-145, which secreted from the gene modified BMSCs, dampened the expression of KLF4. However, the effects of miR-145 on CCSMCs could be rescued by APTO-253. CONCLUSIONS: Overall, miR-145 modification prolongs the retention of the transplanted BMSCs within the CC, and this effect might be attributed to the modulation of the miR-145/KLF4 axis. Consequently, our findings offer a promising and innovative strategy to enhance the local stem cell-based treatments.

Knockdown of programmed cell death factor 4 restores erectile function by attenuating apoptosis in rats with bilateral cavernous nerve crush injury.

BACKGROUND: Apoptosis is an important pathologic mechanism of erectile dysfunction after radical prostatectomy. Studies have shown that programmed cell death factor 4 is connected to the modulation of apoptosis in many cells. However, the programmed cell death factor 4 function in the cavernous nerve injury erectile dysfunction is unclear. OBJECTIVE: This investigation aimed to explore the programmed cell death factor 4 function in erectile dysfunction in rats with bilateral cavernous nerve crush. MATERIALS AND METHODS: The experiment used 30 male Sprague Dawley rats (18 months old) that were screened for normal erectile function by the apomorphine test. Ten rats were randomized into Sham and bilateral cavernous nerve crush groups to detect changes in programmed cell death factor 4 expression. The remaining 20 rats were distributed at random to four groups: the Sham group treated by sham surgery, the phosphate-buffered saline group, the lentivirus containing negative control short hairpin RNA group, and the lentivirus containing short hairpin RNA targeting programmed cell death factor 4 group underwent bilateral cavernous nerve crush and were afterward administered intracavernous injections of phosphate-buffered saline, lentivirus containing negative control short hairpin RNA, or lentivirus containing short hairpin RNA targeting programmed cell death factor 4. Electrical stimulation of the cavernous nerve was conducted 2 weeks later for penile erectile function assessment. The cavernous tissue was collected for histological analysis and western blotting. RESULTS: The apoptosis level in rat corpus cavernosum was elevated, and programmed cell death factor 4 expression was increased after bilateral cavernous nerve crush. Knockdown of programmed cell death factor 4 significantly improved erectile function in bilateral cavernous nerve crush rats. Furthermore, lentivirus containing short hairpin RNA targeting programmed cell death factor 4 treatment raised smooth muscle content and attenuated cavernous fibrosis and apoptotic levels. Additionally, programmed cell death factor 4 was found to mediate the PI3K/AKT pathway. DISCUSSION AND CONCLUSION: Elevated programmed cell death factor 4 expression may be an important pathogenetic mechanism for erectile dysfunction after bilateral cavernous nerve crush, and the knockdown of programmed cell death factor 4 enhanced erectile function in 18-month-old rats after cavernous nerve damage. The potential mechanism may be the stimulation of the PI3K/AKT pathway to attenuate the cavernous apoptosis level.

Caveolin-1 scaffolding domain peptide prevents corpus cavernosum fibrosis and erectile dysfunction in bilateral cavernous nerve injury-induced rats.

BACKGROUND: Corpus cavernosum (CC) fibrosis significantly contributes to post-radical prostatectomy erectile dysfunction (pRP-ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has gained significant concern as a potent antagonist of tissue fibrosis. However, applying CSD peptide on bilateral cavernous nerve injury (BCNI)-induced rats remains uninvestigated. AIM: The aim was to explore the therapeutic outcome and underlying mechanism of CSD peptide for preventing ED in BCNI rats according to the hypothesis that CSD peptide may exert beneficial effects on erectile tissue and function following BCNI through limiting collagen synthesis in CC smooth muscle cells (CCSMCs) and CC fibrosis. METHODS: After completing a random assignment of male Sprague Dawley rats (10 weeks of age), BCNI rats received either saline or CSD peptide treatment, as opposed to sham-operated rats. The evaluations of erectile function (EF) and succedent collection and histological and molecular biological examinations of penile tissue were accomplished 3 weeks postoperatively. In addition, the fibrotic model of CCSMCs was used to further explore the mechanism of CSD peptide action in vitro. OUTCOMES: The assessments of EF, SMC/collagen ratio, α-smooth muscle actin, caveolin-1 (CAV1), and profibrotic indicators expressions were conducted. RESULTS: BCNI rats exhibited significant decreases in EF, SMC/collagen ratio, α-SMA, and CAV1 levels, and increases in collagen content together with transforming growth factor (TGF)-ß1/Smad2 activity. However, impaired EF, activated CC fibrosis, and Smad2 signaling were attenuated after 3 weeks of CSD peptide treatment in BCNI rats. In vitro, TGF-ß1-induced CCSMCs underwent fibrogenetic transformation characterized by lower expression of CAV1, higher collagen composition, and phosphorylation of Smad2; then, the delivery of CSD peptide could significantly block CCSMC fibrosis by inactivating Smad2 signaling. CLINICAL IMPLICATIONS: Based on available evidence of CSD peptide in the prevention of ED in BCNI rats, this study can aid in the development and clinical application of CSD peptide targeting pRP-ED. STRENGTHS AND LIMITATIONS: This study provides data to suggest that CSD peptide protects against BCNI-induced deleterious alterations in EF and CC tissues. However, the available evidence still does not fully clarify the detailed mechanism of action of CSD peptide. CONCLUSION: Administration of CSD peptide significantly retarded collagen synthesis in CCSMCs, limited CC fibrosis, and prevented ED via confrontation of TGF-ß1/Smad signaling in BCNI rats.

An animal model induced by bilateral cavernous nerve crushing mimics post-radical prostatectomy erectile dysfunction in old rats.

AIM: Over the years, the cavernous nerve (CN) crushing injury rat model has been frequently used for studying post-radical prostatectomy erectile dysfunction (pRP-ED). However, models based on young and healthy rats reportedly exhibit spontaneous recovery of erectile function. Our investigation purpose was to evaluate bilateral CN crushing (BCNC) effects on erectile function besides penile corpus cavernosum pathology in young and old rats and verify whether the BCNC modeling in old rats is more suitable to mimic pRP-ED. MATERIALS AND METHODS: Thirty young and old male Sprague-Dawley (SD) rats had been divided into three groups in a random manner: sham-operated group (Sham), CN-injured 2-week group (BCNC-2W), and CN-injured 8-week group (BCNC-8W). At 2 and 8 weeks postoperatively, mean arterial pressure (MAP) along with intracavernosal pressure (ICP) had been determined, respectively. Then, the penis was harvested for histopathological studies. KEY FINDING: We found that young rats exhibited erectile function spontaneous recovery 8 weeks following BCNC, while old ones failed to recover erectile function. After BCNC, the abundance of nNOS-positive nerve and smooth muscle were reduced, whereas apoptotic levels and collagen I content increased. These pathological modifications gradually resumed over time in young rats, unlike in old rats. SIGNIFICANCE: Our findings demonstrate that 18-month-old rats do not spontaneously regain erectile function at 8 weeks after BCNC. Therefore, CN-injury ED modeling in 18-month-old rats may be more suitable for studying pRP-ED.

Heterogeneity of fibroblasts is a hallmark of age-associated erectile dysfunction.

BACKGROUND: The prevalence of age-associated erectile dysfunction (ED) increases pronouncedly with age. However, the cellular composition and transcriptomic changes of aging penile corpus cavernosum remain largely unclear. METHODS: Herein, we performed single cell sequencing penile corpus cavernosum from five young with normal erectile response and five old rats with ED. RESULTS: Clustering analysis identified 19 cell types, such as fibroblasts, myofibroblasts and immune cells. We next revealed their transcriptomic alterations and investigated novel subpopulations of major cell types. Among them, fibroblasts possessed the largest cell number and showed apparent heterogeneity. By performing single-cell entropy analysis on fibroblasts, we observed the age-associated decrease of entropy, and aged fibroblasts were found to adopt senescent secretory phenotype, as evidenced by the high expression of genes associated with the senescence-associated secretory phenotype (SASP). Finally, we constructed a comprehensive intercellular communication network and highlighted key mediators of crosstalk between fibroblasts and other cell types. CONCLUSIONS: We plotted a cellular atlas of aging cells within penile corpus cavernosum, especially fibroblasts. Our work will deepen the understanding of the heterogeneity among certain cell types within aged penile corpus cavernosum, which will generate positive effects on the future treatment of age-associated ED.

Molecular Characterization and Clinical Relevance of N6-Methyladenosine Regulators in Metastatic Prostate Cancer.

Prostate cancer is a leading malignancy in the male population globally. N6-methylation of adenosine (m6A) is the most prevalent mRNA modification and plays an essential role in various biological processes in vivo. However, the potential roles of m6A in metastatic prostate cancer are largely unknown. In this study, we evaluated and identified two m6A modification patterns based on 21 m6A regulators in four public metastatic prostate cancer datasets. Different modification patterns correlated with distinct molecular characteristics. According to m6A-associated genes, we constructed a prognostic model, called m6Ascore, to predict the outcomes of patients with metastatic prostate cancer. We found that high m6A score level was related to dismal prognosis and characterized by higher cell cycle, DNA repair and mismatch repair pathway score. In vitro experiments confirmed that upregulation of METTL14, an m6A writer, enhanced the invasion, metastasis, and sensitivity of prostate cancer cells to poly (ADP-ribose) polymerase inhibitor. Conversely, down-regulation of potential target genes of m6A had the opposite effect. Finally, we validated that a higher m6A score was associated with a worse prognosis and a higher Gleason score in The Cancer Genome Atlas Program (TCGA) cohort. This work illustrated the nonnegligible role of m6A modification in multiple biological processes of metastatic prostate cancer. Evaluating the m6A risk scores of individual tumours will guide more effective judgement of prognosis as well as treatments for metastatic prostate cancer in clinical practice.

Extracellular vesicles derived from mesenchymal stem cells alleviate neuroinflammation and mechanical allodynia in interstitial cystitis rats by inhibiting NLRP3 inflammasome activation.

BACKGROUND: Neuroinflammation in spinal dorsal horn (SDH) plays an important role in the pathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS). Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) exert potent anti-inflammatory activities in the treatment of various diseases. This study aimed to determine the therapeutic effects of MSC-EVs on IC and furtherly investigate the potential mechanism to attenuate neuroinflammation. METHODS: Female IC rat model was established by intraperitoneal injection of cyclophosphamide (50 mg/kg, every 3 days for 3 doses). Inhibition of NLRP3 inflammasome was performed by intraperitoneal injection of MCC950 (10 mg/kg). MSC-EVs were isolated from the culture supernatants of human umbilical cord derived MSCs using ultracentrifugation, and then injected intrathecally into IC rats (20 µg in 10 µl PBS, every other day for 3 doses). Suprapubic mechanical allodynia was assessed using up-down method with von Frey filaments, and micturition frequency was examined by urodynamics. The expression of NLRP3 inflammasome components (NLRP3 and Caspase-1), glial cell markers (IBA-1 and GFAP), proinflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-18) and TLR4/NF-κB signal pathway (TLR4, p65 NK-κB and phospho-p65 NK-κB) in L6-S1 SDH was measured by Western blot analysis. The cellular localization of NLRP3 in SDH was detected using immunofluorescence co-staining. RESULTS: NLRP3 inflammasome was activated in neurons in SDH of IC rats. NLRP3 inflammasome activation contributed to activation of glial cells and process of spinal neuroinflammation in IC rats, and was related to suprapubic mechanical allodynia and frequent micturition. Intrathecal injection of MSC-EVs alleviated suprapubic mechanical allodynia and frequent micturition in IC rats, restrained activation of glial cells and attenuated neuroinflammation in SDH. In addition, MSC-EV treatment significantly inhibited activation of both NLRP3 inflammasomes and TLR4/NF-κB signal pathway. CONCLUSIONS: NLRP3 inflammasome activation is involved in the neuroinflammation of IC. Intrathecal injection of MSC-EVs alleviates neuroinflammation and mechanical allodynia in IC by inhibiting the activation of NLRP3 inflammasome, and TLR4/NF-κB signal pathway may be the potential regulatory target.

Upregulated IGFBP3 with Aging Is Involved in Modulating Apoptosis, Oxidative Stress, and Fibrosis: A Target of Age-Related Erectile Dysfunction.

Aging has been deemed the primary factor in erectile dysfunction (ED). Herein, age-related changes in the erectile response and histomorphology were detected, and the relationship between aging and ED was investigated based on gene expression levels. Thirty male Sprague-Dawley (SD) rats were randomly divided into 6 groups, and intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured. Subsequently, the corpus cavernosum (CC) was harvested and prepared for histological examinations of apoptosis, oxidative stress (OS), and fibrosis. Then, the microarray dataset (GSE10804) was analyzed to identify differentially expressed genes (DEGs) in ED progression, and hub genes were selected. In addition, aged CC smooth muscle cells (CCSMCs) were isolated to evaluate the function of the hub gene by siRNA interference, qRT-PCR, immunofluorescence staining, enzyme-linked immunosorbent assay, western blot analysis, CCK-8 assay, EdU staining, and flow cytometry approaches. The ICP/MAP and smooth muscle cell (SMC)/collagen ratios declined with aging, while apoptosis and OS levels increased with aging. The enriched functions and pathways of the DEGs were investigated, and 15 hub genes were identified, among which IGFBP3 was significantly upregulated. The IGFBP3 upregulation was verified in the CC of aging rats. Furthermore, aged CCSMCs were transfected with siRNA to knock down IGFBP3 expression. The viability and proliferation of the CCSMCs increased, while apoptosis, OS, and fibrosis decreased. Our findings demonstrate that the erectile response of SD rats declines in parallel with enhanced CC apoptosis, OS, and fibrosis with aging. Upregulation of IGFBP3 plays an important role; furthermore, downregulation of IGFBP3 improves the viability and proliferation of CCSMCs and alleviates apoptosis, OS, and fibrosis. Thus, IGFBP3 is a potential therapeutic target for age-related ED.

Notch1 Signaling Contributes to Mechanical Allodynia Associated with Cyclophosphamide-Induced Cystitis by Promoting Microglia Activation and Neuroinflammation.

AIMS: Notch1 signaling regulates microglia activation, which promotes neuroinflammation. Neuroinflammation plays an essential role in various kinds of pain sensation, including bladder-related pain in bladder pain syndrome/interstitial cystitis (BPS/IC). However, the impact of Notch1 signaling on mechanical allodynia in cyclophosphamide- (CYP-) induced cystitis is unclear. This study is aimed at determining whether and how Notch1 signaling modulates mechanical allodynia of CYP-induced cystitis. METHODS: CYP was peritoneally injected to establish a bladder pain syndrome/interstitial cystitis (BPS/IC) rat model. A γ-secretase inhibitor, DAPT, was intrathecally injected to modulate Notch1 signaling indirectly. Mechanical withdrawal threshold in the lower abdomen was measured with von Frey filaments using the up-down method. The expression of Notch1 signaling, Iba-1, OX-42, TNF-α, and IL-1ß in the L6-S1 spinal dorsal horn (SDH) was measured with Western blotting analysis and immunofluorescence staining. RESULTS: Notch1 and Notch intracellular domain (NICD) were both upregulated in the SDH of the cystitis group. Moreover, the expression of Notch1 and NICD was negatively correlated with the mechanical withdrawal threshold of the cystitis rats. Furthermore, treatment with DAPT attenuated mechanical allodynia in CYP-induced cystitis and inhibited microglia activation, leading to decreased production of TNF-α and IL-1ß. CONCLUSION: Notch1 signaling contributes to mechanical allodynia associated with CYP-induced cystitis by promoting microglia activation and neuroinflammation. Our study showed that inhibition of Notch1 signaling might have therapeutic value for treating pain symptoms in BPS/IC.

Methylcrotonoyl-CoA Carboxylase 2 Promotes Proliferation, Migration and Invasion and Inhibits Apoptosis of Prostate Cancer Cells Through Regulating GLUD1-P38 MAPK Signaling Pathway.

PURPOSE: Prostate cancer (PCa) is the most common cancer in American men, and the mechanisms of development and progression are still not completely clear. Methylcrotonoyl-CoA carboxylase 2 (MCCC2) was previously identified overexpressed in PCa with lymph node metastasis, but its specific role and mechanisms need further investigation. This study aimed to investigate the role of MCCC2 in PCa cells and its underlying mechanisms. MATERIALS AND METHODS: Quantitative RT-PCR and Western blotting were used to detect MCCC2 mRNA and protein expression in normal prostate epithelium and cancerous cells. Upon manipulation of MCCC2 expression, cell proliferation was measured by CCK-8 assays and migration and invasion were determined by transwell assays. Changes of apoptosis, cell cycle and mitochondrial membrane potential were evaluated by flow cytometry. MCCC2-mediated signaling pathways were screened by bioinformatics and verified by RT-PCR and Western blotting. Finally, immunohistochemistry was performed to detect the expression of MCCC2 and glutamate dehydrogenase 1 (GLUD1) in PCa tissues to analyze their correlation. RESULTS: We demonstrated that MCCC2 promoted cell proliferation, migration and invasion but inhibited apoptosis in PCa cells. In addition, MCCC2 in 22Rv1 cells induced mitochondrial damage. In PCa tissues, MCCC2 overexpression associated with lymph node metastasis (P=0.001) and high Gleason scores (P<0.001). MCCC2 positively correlated with GLUD1 expression in PCa tissues (r=0.435, P<0.001). Ectopic overexpression of MCCC2 up-regulated GLUD1 and p38 MAPK expression, whereas inhibition of MCCC2 decreased GLUD1 and p38 MAPK expression. CONCLUSION: MCCC2 exerts oncogenic function in PCa through regulating GLUD1-p38 MAPK signaling pathway, and it may be a potential treatment target.

Proteomic analysis of oxidative stress response in human umbilical vein endothelial cells (HUVECs): role of heme oxygenase 1 (HMOX1) in hypoxanthine-induced oxidative stress in HUVECs.

BACKGROUND: Erectile dysfunction (ED) is a well-known complication of diabetes, affecting up to 75% of diabetic men. Although the etiology of diabetic ED is multifactorial, endothelial dysfunction is considered to be a pillar of its pathophysiology. Endothelial dysfunction is caused by the harmful effects of high glucose levels and increased oxidative stress on the endothelial cells that comprise the vascular endothelium. The aim of this study was to identify the proteomic changes caused by high glucose-induced oxidative stress and explore the role of heme oxygenase 1 (HMOX1) in it. METHODS: The cellular proteomic response to hypoxanthine-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were analyzed through Network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Further validation assays was performed to validate the role of HMOX1. RESULTS: The results showed that 66 and 76 DEPs were markedly upregulated and downregulated, respectively, for HUVECs oxidative stress. Among these proteins, we verified eight dysregulated genes by quantitative reverse transcription PCR, including nucleolin (NCL), X-ray repair cross-complementing protein 6 (XRCC6), ubiquinol-cytochrome C reductase binding protein (UQCRB), non-POU domain containing octamer binding (NONO), heme oxygenase 1 (HMOX1), nucleobindin 1 (NUCB1), DEK, and chromatin target of prmt1 (CHTOP). Further, using overexpression and genetic knockdown approaches, we found that HMOX1 was critical for the oxidative stress response in HUVECs. CONCLUSIONS: We found that HMOX1 was closely related to the oxidative stress response induced by hypoxanthine. To the best of our knowledge, this study is the first overview of the responses of HUVECs to oxidative stress. The findings will contribute to analyses of the detailed molecular mechanisms involved in the pathogenesis of endothelial dysfunction and related molecular mechanisms in ED patients.

MicroRNA-145 engineered bone marrow-derived mesenchymal stem cells alleviated erectile dysfunction in aged rats.

BACKGROUND: Aging is one of the dominant factors contributing to erectile dysfunction (ED), and effective treatments for age-associated ED are urgently demanded. In this study, the therapeutic efficiency of bone marrow-derived mesenchymal stem cells (BMSCs) overexpressing microRNA-145 (miR-145) was evaluated in ED. METHODS: Sixty male Sprague-Dawley rats (24 months old) were randomly divided into 4 treatment groups (n = 15/group): PBS (control), BMSCs, BMSCs transfected with a blank vector (vector-BMSCs), and BMSCs transfected with a lentivirus overexpressing miR-145 (OE-miR-145-BMSCs). Fourteen days after transplantation of BMSCs, erectile function was evaluated by measuring intra-cavernous pressure (ICP) and mean arterial pressure (MAP). Subsequently, penile erectile tissues were harvested and subjected to Masson staining, qRT-PCR, immunofluorescence staining, dual luciferase assay, and Western blot analysis. RESULTS: Fourteen days after transplantation, the ICP/MAP was 0.79 ± 0.05 in the OE-miR-145-BMSC group, 0.61 ± 0.06 in the BMSC group, 0.57 ± 0.06 in the vector-BMSC group, and 0.3 ± 0.01 in the PBS group. Treatment with OE-miR-145-BMSCs significantly improved ED (P < 0.05), and the treatment increased the smooth muscle content in the penis tissues of ED rats (P < 0.05). In the OE-miR-145-BMSC group, the expression levels of α-SMA, desmin, and SM-MHC were higher than they were in the other ED groups (P < 0.05). In addition, the levels of collagen 1, MMP2, and p-Smad2 in the BMSC-treated group, especially in the OE-miR-145-BMSC group, were lower than those in the control group (P < 0.05). CONCLUSIONS: MicroRNA-145 engineered BMSCs effectively attenuate age-related ED. Transplantation of miR-145-overexpressing BMSCs may provide a promising novel avenue for age-associated ED therapy.

GLUT1 regulates cell glycolysis and proliferation in prostate cancer.

BACKGROUND: Glucose transporter 1 (GLUT1) plays a critical role in tumorigenesis and tumor progression in multiple cancer types. However, the specific function and clinical significance of GLUT1 in prostate cancer (PCa) are still unclear. Therefore, in this study, we investigated the role of GLUT1 in PCa. METHODS: GLUT1 protein levels in prostate cancer tissue and tumor-adjacent normal tissues were measured and compared. Furthermore, real-time PCR and Western blot analysis were both used to detect GLUT1 expression levels in different PCa cell lines. Flow cytometry and cell-based assays, such as a glucose uptake and lactate secretion assay, CCK-8 assay, and transwell migration and wound healing assay, were used to monitor cancer cell cycle distribution, glycolysis, proliferation, and motility, respectively. Moreover, a mouse tumor xenograft model was used to investigate the role of GLUT1 in tumor progression in vivo. RESULTS: GLUT1 expression levels are higher in PCa tissues than in tumor-adjacent normal tissues. The results from real-time PCR and Western blot analysis revealed a similar increase in the GLUT1 expression levels in PCa cell lines. Moreover, knockdown of GLUT1 inhibits cell glycolysis and proliferation and leads to cell cycle arrest at G2/M phase in the 22RV1 cell line but not in the PC3 cell line. In vivo experiments further confirmed that GLUT1 knockdown inhibits the growth of tumors derived from the 22RV1 cell line. In addition, we also showed that GLUT1 knockdown has no effect on cell migration in vitro. CONCLUSIONS: GLUT1 may play an important role in PCa progression via mediating glycolysis and proliferation. Our study also indicated a potential crosstalk between GLUT1-mediated glycolysis and androgen sensitivity in PCa.

Increased expression of glycolytic enzymes in prostate cancer tissues and association with Gleason scores.

OBJECTIVE: Recent studies have shown that understanding the differences between Gleason 3+4 and Gleason 4+3 in PCa patients may improve their treatment. This study aimed to evaluate the different expression levels of glycolytic proteins for Gleason score of 4+3 and 3+4. METHODS: A total of 90 PCa patients, including 38 cases with a Gleason score of 7, were included in this study. The expression of glycolytic proteins in both prostate cancer and normal prostate tissues, in GGG2 and GGG3 as well were assessed by immunohistochemical staining. RESULTS: Compared with GGG3, the GGG2 cases displayed significantly lower expression of all proteins (P < 0.05). The correlation among all enzymes showed that the key glycolytic enzyme, HK2, was significantly positively related to another key enzyme, PKM2 (r = 0.550, P < 0.01), and the expression of PFKFB4 was correlated with the expression of HK2 (r = 0.236, P < 0.05) and PKM2 (r = 0.392, P < 0.01). Additionally, neither GLUT1 nor PFKFB3 was correlated with PFKFB4, HK2 or PKM2. Further analysis showed that HK2 (r = 0.297, P < 0.01) and PKM2 (r = 0.431, P < 0.01) were significantly positively related to the Gleason score in PCa tissues. CONCLUSIONS: Glycolytic proteins expression levels were upregulated in PCa tissues. Furthermore, GGG3 exhibits a higher level of glycolysis compared with GGG2 in PCa tissues. Additionally, the key glycolytic enzymes, HK2 and PKM2, are overexpressed simultaneously in PCa and significantly correlate with PCa progression as represented by the GS.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase.

Prostate cancer is the most commonly diagnosed non-cutaneous cancer and one of the leading causes of cancer death for North American men. Whereas localized prostate cancer can be cured, there is currently no cure for metastatic prostate cancer. Here we report a novel approach that utilizes designed chimeric transcription activator-like effectors (dTALEs) to control prostate cancer metastasis. Transfection of dTALEs of DNA methyltransferase or demethylase induced artificial, yet active locus-specific CpG and subsequent histone modifications. These manipulations markedly altered expression of endogenous CRMP4, a metastasis suppressor gene. Remarkably, locus-specific CpG demethylation of the CRMP4 promoter in metastatic PC3 cells abolished metastasis, whereas locus-specific CpG methylation of the promoter in non-metastatic 22Rv1 cells induced metastasis. CRMP4-mediated metastasis suppression was found to require activation of Akt/Rac1 signaling and down-regulation of MMP-9 expression. This proof-of-concept study with dTALEs for locus-specific epigenomic manipulation validates the selected CpG methylation of CRMP4 gene as an independent biomarker for diagnosis and prognosis of prostate cancer metastasis and opens up a novel avenue for mechanistic research on cancer biology.

Rare earths exposure and male infertility: the injury mechanism study of rare earths on male mice and human sperm.

The weight; testis/body coefficient; levels of LDH, SDH, SODH, G-6PD, and testosterone; cell cycle; and cell apoptosis of the male mice were influenced after being treated with 200 mg/[kg/day] of rare earths suspension for 3 weeks. The "Raman fingerprints" of the human sperm DNA exposed to 0.040 mg/ml CeCl3 were very different from those of the untreated; the Raman bands at 789 cm(-1) (backbone phosphodiester), PO4 backbone at 1,094 cm(-1), methylene deformation mode at 1,221 cm(-1), methylene deformation mode at 1,485 cm(-1), and amide II at 1,612 cm(-1), of which intensities and shifts were changed, might be the diagnostic biomarkers or potential therapeutic targets. The injury mechanism might be that the rare earths influence the oxidative stress and blood testosterone barrier, tangle the big biomolecule concurrently, which might cause the testicular cells and vascular system disorder and/or dysfunction, and at the same time change the physical and chemical properties of the sperm directly.