Dopamine as a potential diagnostic biomarker in women's sexual dysfunction.

Dopamine and prolactin are the key mediators involved in sexual function in both males and females, but the role of dopamine in female sexual dysfunction (FSD) is still unclear. The aim was to investigate the possible role of dopamine and their relationship with sex steroid hormones (estrogen, progesterone and dehydroepiandrosterone; DHEA) and prolactin levels in Egyptian women suffering from sexual dysfunction. This study included 84 women having sexual dysfunction (FSD group) and 84 normal sexual function (control group). All women were subjected to the questionnaire to assess their demographic and gynecological data as well as female sexual function index (FSFI). Blood samples were collected from all women for measuring serum estradiol, progesterone, DHEA, prolactin and dopamine levels. FSD patients had significantly higher serum progesterone and DHEA and prolactin levels; while significantly lower dopamine and estradiol levels versus controls (p < 0.001). In all women, dopamine level appeared as a predictor of FSD at cut-off point ≤8.8 ng/mL with sensitivity (75%), specificity (92%) and accuracy (83%) (p < 0.001). The low levels of dopamine were associated with significantly higher prevalence in patients with low estradiol (p < 0.001) and high progesterone (p < 0.001), DHEA (p < 0.001) and prolactin (p = 0.004). Also, dopamine was significantly positive correlation with arousal score (r = 0.16, p = 0.04), and negative correlation with age (r = -0.31, p < 0.001), pain score (r = -0.19, p = 0.01), DHEA (r = -0.45, p < 0.001) and prolactin (r = -0.28, p < 0.001). Low serum dopamine level is a potential diagnostic biomarker in women's sexual dysfunction and their association with high prolactin and sex steroid hormones dysfunction.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

Design and cytotoxic evaluation via apoptotic and antiproliferative activity for novel 11(4-aminophenylamino)neocryptolepine on hepatocellular and colorectal cancer cells.

The current study evaluated the cytotoxic activity of 11(4-Aminophenylamino)neocryptolepine (APAN), a novel derivative of neocryptolepine, on hepatocellular (HepG2) and colon (HCT-116) carcinoma cell lines as well as, the possible molecular mechanism through which it exerts its cytotoxic activity. The APAN was synthesized and characterized based on their spectral analyses. Scanning for anticancer target of APAN by Swiss software indicated that APAN had highest affinity for protein tyrosine kinase 6 enzyme. Furthermore, Super pred software indicated that APAN can be indicated in hepatic and colorectal cells with 92%. Molecular docking studies indicated that the binding affinity scores of APAN for protein PDB code: 6CZ4 of tyrosine kinase 6 recorded of - 6.6084 and RMSD value of 0.8891°A, while that for protein PDB: 7JL7 of caspase 3 was - 6.1712 and RMSD of 0.8490°A. Treatment of HepG2 and HCT-116 cells with APAN induced cytotoxicity with IC50 of 2.6 and 1.82 µg/mL respectively. In addition, it induced injury and serious morphological changes in cells including, disappearance of microvilli, membrane blebbing, cytoplasmic condensation, and shrunken nucleus with more condensed chromatin. Moreover, APAN significantly increased protein expression of annexin V (apoptotic marker). Furthermore, APAN significantly increased protein expression of caspase 3 and P53. However, it significantly reduced secretion of VEGF protein into the medium and decreased protein expression of PCNA and Ki67 in HepG2 and HCT-116 cells. This study indicated that APAN had cytotoxic activity against HepG2 and HCT-116 cells via increasing the expression of apoptotic proteins and reducing the expression of proliferative proteins.

Serum cystatin C is an early renal dysfunction biomarker in patients with hepatitis C virus.

Background: Hepatitis C virus (HCV) may induce extrahepatic manifestations as acute or chronic renal dysfunction. The aim was to evaluate the diagnostic role of some biomarkers as cystatin C, cryoglobulins, rheumatoid factor (RF), and complement C3 for extrahepatic renal affection in newly diagnosed patients with HCV infection. Methods: Blood and urine were collected from randomized individuals screened for new HCV infection (n=400). The studied populations were divided into 3 groups: control group I: thirty healthy individuals not suffering from either liver or kidney diseases, group IIa: thirty HCV patients who have positive HCV antibody test but showed negative PCR test, and group IIb: thirty HCV patients who showed positive results for both HCV antibody and PCR tests. Results: In HCV group IIb, levels of serum total bilirubin, AST and ALT, and urine albumin/creatinine ratio were increased whereas serum albumin and creatinine clearance were decreased versus other groups. However, the levels of blood urea nitrogen and serum creatinine were still within the normal range in all groups. In HCV group IIb, cystatin C, cryoglobulins, and RF levels were increased; meanwhile, serum creatinine/cystatin C ratio and complement 3 levels were decreased compared to the other groups. HCV-infected patients significantly had higher serum cystatin C (>1.24 mg/L, P<0.001) and lower creatinine/cystatin C ratio (<70.1µMol/mg, P=0.002), and cystatin C was significantly correlated with liver and kidney parameters. Conclusion: High serum cystatin C and low creatinine/cystatin C ratio may be early indicators of mild renal dysfunction with normal serum levels of creatinine in HCV-infected individuals.

Potential impact of serpin peptidase inhibitor clade (A) member 4 SERPINA4 (rs2093266) and SERPINA5 (rs1955656) genetic variants on COVID-19 induced acute kidney injury.

Background: SARS-CoV-2 has a number of targets, including the kidneys. Acute Kidney Injury (AKI) might develop in up to a quarter of SARS-CoV-2 patients. In the clinical environment, AKI is linked to a high rate of death and leads to the progression of AKI to chronic renal disease. Aim: We aimed to investigate rs2093266 and rs1955656 polymorphisms in SERPINA4 and SERPINA5 genes, respectively, as risk factors for COVID-19 induced AKI. Subjects and methods: A case-control study included 227 participants who were divided into three groups: 81 healthy volunteers who served as controls, 76 COVID-19 patients without AKI and 70 COVID -19 patients with AKI. The TaqMan assay was used for genotyping the SERPINA4 (rs2093266) and SERPINA5 (rs1955656) polymorphisms by real-time PCR technique. Results: Lymphocytes and eGFR showed a significantly decreasing trend across the three studied groups, while CRP, d-Dimer, ferritin, creatinine, KIM-1and NGAL showed a significantly increasing trend across the three studied groups (P < 0.001). Rs2093266 (AG and AA) genotypes were significant risk factors among non-AKI and AKI groups in comparison to controls. Rs1955656 (AG and AA) were significant risk factors among the AKI group, while AA was the only significant risk factor among the non-AKI group. Recessive, dominant, co-dominant, and over-dominant models for genotype combinations were demonstrated. The GG v AA, GG + AG v AA, and GG v AG + AA models of the rs2093266 were all significant predictors of AKI, whilst only the GG v AA model of the rs1955656 SNP was a significant predictor. The logistic regression model was statistically significant, χ2 = 56.48, p < 0.001. AKI was associated with progressed age (OR = 0.95, 95% CI: 0.91-0.98, p = 0.006), suffering from chronic diseases (OR = 3.25, 95% CI: 1.31-8.01, p = 0.010), increased BMI (OR = 0.89, 95% CI: 0.81-0.98, p = 0.018), immunosuppressive (OR = 4.61, 95% CI: 1.24-17.16, p = 0.022) and rs2093266 (AG + AA) (OR = 3.0, 95% CI: 1.11-8.10, p = 0.030). Conclusion: Single nucleotide polymorphisms (rs2093266) at SERPINA4 gene and (rs1955656) at SERPINA5 gene were strongly linked to the development of AKI in COVID-19 patients.

BMP-7 Attenuates Inflammation-Induced Pyroptosis and Improves Cardiac Repair in Diabetic Cardiomyopathy.

In the present study, we investigated a novel signaling target in diabetic cardiomyopathy where inflammation induces caspase-1-dependent cell death, pyroptosis, involving Nek7-GBP5 activators to activate the NLRP3 inflammasome, destabilizes cardiac structure and neovascularization. Furthermore, we explored the therapeutic ability of bone morphogenetic protein-7 (BMP-7) to attenuate these adverse effects. C57BL/6J mice (n = 16 mice/group) were divided into: control (200 mg/kg, 0.9% saline intraperitoneal injection, i.p.); Streptozotocin (STZ) and STZ-BMP-7 groups (STZ, 200 mg/kg, i.p. injection). After 6 weeks, heart function was examined with echocardiography, and mice were sacrificed. Immunostaining, Western blotting, H&E, and Masson's trichrome staining was performed on heart tissues. STZ-induced diabetic cardiomyopathy significantly increased inflammasome formation (TLR4, NLRP3, Nek7, and GBP5), pyroptosis markers (caspase-1, IL-1ß, and IL-18), inflammatory cytokines (IL-6 and TNF-α), MMP9, and infiltration of monocytes (CD14), macrophage (iNOS), and dendritic cells (CD11b and CD11c) (p < 0.05). Moreover, a significant endothelial progenitor cells (EPCs) dysfunction (c-Kit/FLk-1, CD31), adverse cardiac remodeling, and reduction in left ventricular (LV) heart function were observed in STZ versus control (p < 0.05). Treatment with BMP-7 significantly reduced inflammasome formation, pyroptosis, and inflammatory cytokines and infiltrated inflammatory cells. In addition, BMP-7 treatment enhanced EPC markers and neovascularization and subsequently improved cardiac remodeling in a diabetic heart. Moreover, a significant improvement in LV heart function was achieved after BMP-7 administration relative to diabetic mice (p < 0.05). In conclusion, BMP-7 attenuated inflammation-induced pyroptosis, adverse cardiac remodeling, and improved heart function via the TLR4-NLRP3 inflammasome complex activated by novel signaling Nek7/GBP5. Our BMP-7 pre-clinical studies of mice could have significant potential as a future therapy for diabetic patients.

Assessment of temporal functional changes and miRNA profiling of human iPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.

Association between cataract progression and ischemia-modified albumin in relation to oxidant-antioxidant profiles in the serum, aqueous humor, and lens.

PURPOSE: To evaluate the levels of ischemia-modified albumin (IMA) in relation to oxidant-antioxidant profiles in the serum, aqueous, and lens in cataract patients. SETTING: Department of Ophthalmology, Menoufia University, Shebin El Kom, Menoufia, Egypt. DESIGN: Prospective case series. METHODS: Patients were divided into 2 groups. The cataract (study) group comprised patients with senile cataract and the control group, age- and sex-matched healthy persons. Patients with systemic disease or cataract formation secondary to identifiable causes were excluded. In all cases, a complete history was taken and a clinical examination was performed. In the cataract group, the lens was examined, and the cataract type and severity were graded. Blood levels of catalase, malondialdehyde (MDA), superoxide dismutase (SOD), and IMA were measured in all participants and in the aqueous and lens lysate of cataract patients. RESULTS: Each group comprised 30 participants. Cataract patients had significant higher levels of serum MDA and IMA than the control group but had lower levels of serum catalase and SOD. Patients with cortical cataracts had higher level of serum IMA, aqueous catalase, and SOD levels patients with nuclear cataracts but had a lower level of lens SOD. There was a significant positive correlation between serum MDA and the patient's age and serum catalase levels. CONCLUSION: Patients with cortical cataract had increased local oxidative stress and diminished antioxidant activity compared with systemic oxidative activity, which was not the same in patients with nuclear cataract.

Tadalafil, a long acting phosphodiesterase inhibitor, promotes bone marrow stem cell survival and their homing into ischemic myocardium for cardiac repair.

The aim was to evaluate the tadalafil-mediated effects at molecular level on bone marrow-derived mesenchymal stem cells (MSCs) survival and their homing into the infarcted hearts to promote cardiac repair and improve function. MSCs were pretreated in vitro with inhibitors of PKG, MAPK, FasL, nitric oxide synthase (NOS) (L-NAME), CXCR4 (AMD3100), or miR-21 inhibitors (+/-luciferase construction +/-Fas) prior to tadalafil treatment for 2 h. These MSCs were then subjected to H2O2 stress to assess their injury. Rats were subjected to acute myocardial infarction (AMI), and then followed by injection of saline or 1.5 x 106 MSCs-treated ± tadalafil into infarcted and peri-infarcted area. In another group, AMI was performed in 1-month post-myelo-ablated rats and were injected intraperitoneally (IP) with tadalafil ± AMD3100 or L-NAME for 5 days. Also, in another group, AMI mice were treated with IP ± tadalafil before intravenous injection with 111In-oxine-MSCs followed by CT/SPECT imaging to locate mobilized MSCs. Cardiac function was assessed by echocardiography. MSCs and heart extracts were analyzed by molecular bioassays. Tadalafil-treated MSCs had higher expression of cGMP, NOS, SDF-1α, p-VASP, p-Erk1/2, p-STAT3, p-Akt, PKG1 and Bcl-xl; expression of these molecules was reduced with PKG1, MAPK, NOS or FasL inhibitors. Tadalafil inhibited apoptosis through increased miR-21 expression and improved cell survival by inhibiting Fas (restored by PKG1, MAPK or miR-21 inhibitors). In vivo, heart function, grafted cell survival, MSCs mobilization and homing were improved in tadalafil-treated AMI animals versus controls. CONCLUSIONS: Tadalafil prolonged MSCs survival via up-regulation of miR-21 dependent suppression of Fas, and increased MSCs mobilization and their homing into infarcted myocardium resulting in improved cardiac repair and function.

Evaluation of Vitamin D, Vitamin D Binding Protein Gene Polymorphism with Oxidant - Antioxidant Profiles in Chronic Obstructive Pulmonary Disease.

BACKGROUND: The aim was to evaluate the association of plasma 25-hydroxyvitamin D (25-OHD) and vitamin D binding protein (VDBP or Gc-globin) gene polymorphism with oxidant-antioxidant profiles in patients with chronic obstructive pulmonary disease (COPD), and their role as biomarker risk factors in susceptibility and severity of COPD. METHODS: Eighty patients diagnosed with COPD (mild, moderate and severe according to lung function tests; FEV 1%) and 80 healthy controls were included in the study. Serum nitric oxide (NO) and lipid peroxide (LP), plasma reduced glutathione (RGSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activity, 25-OHD and VDBP polymorphism were analyzed in all subjects. RESULTS: COPD patients had significantly decreased serum NO, plasma SOD, RGSH, GSH-Px, CAT and 25-OHD versus controls, but had significantly increased serum LP. In COPD patients, 25-OHD levels were significantly lower (41.49± 13.65 ng/mL) versus controls, but more lower in severe COPD patients (30.54±9.09 ng/mL; sensitivity 79.2%; spe - cificity 73.2%, p<0.001) versus mild and moderate COPD. VDBP genotypes frequencies were Gc1S-1S=23.8%, Gc1F-1S=28.8%, Gc1F-1F=15%, Gc1S-2=20%, Gc1F-2=11.3% and Gc2-2=1.3%. Also, VDBP variants frequencies were Gc1S=48.1%, Gc1F=35% and Gc2=16.6%. How ever, Gc1F-1S genotypes and Gc1F variants were significantly higher than in controls (10%, 12.5%; p=0.009, p=0.001, respectively). Moreover, in severe COPD patients, Gc1F-1S genotype was significantly higher than in mild COPD (41.7% vs 31.3%, p=0.04). CONCLUSION: COPD patients had significantly lower plasma 25-OHD and were associated with significantly higher VDBP Gc1F-1S genotypes and Gc1F variants frequencies than controls. Low vitamin D levels and VDBP polymorphism may be important as diagnostic risk factors in the susceptibility to and severity of COPD.

The value of urotensin II in patients with left-sided rheumatic valvular regurgitation.

AIMS: Rheumatic valve diseases are most common etiological valve diseases in developing countries. Urotensin II is cardiovascular autacoid/hormone and may be associated with patients of heart valve diseases. The present study was to measure plasma urotensin II concentrations in patients with left-sided rheumatic valve diseases such as mitral regurgitation (MR) and aortic regurgitation (AR), and to examine its correlation with severity of valve impairment, function (New York Heart association, NYHA) class and pulmonary artery pressure (PAP). METHODS AND RESULTS: Sixty patients with moderate to severe rheumatic left-sided valve regurgitation and 20 healthy controls were selected after performing the echocardiography. Plasma urotensin II level was measured in all subjects. The patients with MR and AR were significantly increased of left ventricular end diastolic dimension (LVEDD), left ventricular end systolic dimension (LVESD), left atrial diameter, PAP, but decreased of EF% versus the controls. Urotensin II level was highly significant in patients with MR (1.83 ± 0.92 ng/ml, P < 0.001) and AR (0.79 ± 0.3 ng/ml, P < 0.05) versus the controls (0.48 ± 0.13 ng/ml). Also, there was significant correlation between Urotensin II level and LVEDD (MR, r = 0.318, P = 0.03; AR, r = 0.805, P < 0.001), LVESD (MR, r = -0.271, P = 0.115; AR, r = 0.614, P = 0.001), and PAP (MR, r = 0.706, P < 0.001; AR, r = 0.129, P = 0.538). CONCLUSION: Urotensin II was elevated in patients with rheumatic left-sided valvular regurgitation, and positively correlated with increased LVEDD (in both MR and AR), LVESD (only AR) and pulmonary artery pressure (only MR). Therefore, urotensin II level may be used as diagnostic biomarker in patients with rheumatic valvular diseases for assessment of the severity in parallel with echocardiography.

Therapeutic potential of fucoidan in myocardial ischemia.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, is a candidate for the treatment of ischemic diseases. The aim of this study was to measure the therapeutic potential of fucoidan in a rat model of myocardial ischemia-reperfusion injury. Forty rats were submitted to myocardial ischemia-reperfusion injury by transient occlusion of the left coronary artery. Rats were then randomized into 2 groups: fucoidan (5 mg/kg, intramuscularly; n = 20) or control (saline intramuscularly; n = 20) was administered 1 hour before injury and daily thereafter for 1 month. At 1 month, plasma levels of stromal cell-derived factor-1α (SDF-1α) were assessed by enzyme-linked immunosorbent assay kit. Hearts were evaluated by histoimmunochemistry. Fucoidan induced significant antifibrotic effects, reducing the infarct scar size by almost 30% on Sirius red-stained sections (9.45% ± 4.27% vs. 13% ± 5.67% in controls; P = 0.03). Vascular density in the fucoidan group (α-actin, RECA-1, or lectin BS1 stained) was increased by 40% (2.18 ± 0.79 mm vs. 1.49 ± 0.42 mm in controls ×200; P = 0.001). Plasma SDF-1α at 1 month was not significantly different between the 2 groups. However, increased immunostaining density of SDF-1α and vascular endothelial growth factor in fibrotic ischemic tissues was observed in fucoidan-treated animals versus controls. In conclusion, fucoidan enhanced tissue repair in myocardial ischemia-reperfusion by promoting revascularization (in situ vascular endothelial growth factor and SDF-1α overexpression) and limiting fibrosis. Consequently, fucoidan may be useful for myocardial ischemic patients.

Preconditioning of Human Skeletal Myoblast with Stromal Cell-derived Factor-1α Promotes Cytoprotective Effects against Oxidative and Anoxic Stress.

BACKGROUND AND OBJECTIVES: The incidence of human autologous transplanted skeletal myoblast (SkM) cell death in ischemic myocardium was higher in the first few days after cell therapy. We proposed that human SkM treated by human stromal cell-derived factor (SDF-1α) protein or tranfected by SDF-1α, precondition them against oxidative or anoxic injury. METHODS AND RESULTS: The purification of human SkM (80∼90%) culture was assessed for desmin and CXCR4 expression using immunostaining and flow cytometry respectively. Cells were transfected to overexpress SDF-1α or treated with rSDF-1α (10∼200 ng/ml, 1∼4 h) were either exposed to anoxia or treated with 100µM H2O2 for different time periods (1∼6 h anoxia) (1∼3 h H2O2). Optimized conditions for transfection of SDF-1α gene into human SkM were achieved, using FuGene(TM)6/phSDF-1α(3：2 v/w, 4 h transfection) with 125µ M ZnCl2 (p＜ 0.001), up to 7 days post-transfection as compared with transfected SkM without ZnCl2 and non-transfected control cells. Transfection efficiency was assessed by immunostaining, ELISA, western blots and PCR. LDH analysis showed significant decrease in release of LDH after exposure to 6 h anoxia or 100µ M H2O2 for 2 h as compared with the normal un-treated or un-transfected SkM (p＜ 0.001). In western blots assay, SDF-1α over-expressing human SkM or treated with rSDF-1α induced marked expression of total Akt (1.2-fold) and phospho-Akt (2.7-fold), Bcl2 (1.6-fold) and VEGF (5.8-fold) after exposure to 6 h anoxia as compared with human SkM controls. CONCLUSIONS: The preconditioning of donor transplanted human SkM with SDF-1α increased cell survival and promoted cytoprotective effect against oxidative or anoxic injury that may be an innovative approach for clinical application.

Mesothelial cells vs. skeletal myoblasts for myocardial infarction.

Cell transplantation for the regeneration of ischemic myocardium is limited by poor graft viability and low cell retention. Omental flaps in association with growth factors and cell sheets have recently been used to increase the vascularization of ischemic hearts. This experimental study was undertaken to evaluate the hemodynamic evolution and histological modifications of infarcted myocardium treated with mesothelial cells, and to compare the results with those of hearts treated with skeletal myoblasts. Myocardial infarction was created by surgical ligature of 2 coronary branches in 34 sheep; 6 died immediately due to ventricular fibrillation. Mesothelial cells were isolated from greater omentum, and myoblasts from skeletal muscle. After expanding the cells for 3 weeks, infarcted areas were treated with culture medium (control group), mesothelial cells, or myoblasts. After 3 months, echocardiographic studies showed significant limitation of ventricular dilatation and improved ejection fractions in both cell-treated groups compared to the controls. In the mesothelial cell group, histological studies showed significantly more angiogenesis and arteriogenesis than in the control and skeletal myoblast groups. Mesothelial cells might be useful for biological revascularization in patients with ischemic heart disease.

Nonviral vector gene modification of stem cells for myocardial repair.

Therapeutic angiogenesis and myogenesis restore perfusion of ischemic myocardium and improve left ventricular contractility. These therapeutic modalities must be considered as complementary rather than competing to exploit their advantages for optimal beneficial effects. The resistant nature of cardiomyocytes to gene transfection can be overcome by ex vivo delivery of therapeutic genes to the heart using genetically modified stem cells. This review article gives an overview of different vectors and delivery systems in general used for therapeutic gene delivery to the heart and provides a critical appreciation of the ex vivo gene delivery approach using genetically modified stem cells to achieve angiomyogenesis for the treatment of infarcted heart.

Mesothelial cell transplantation in the infarct scar induces neovascularization and improves heart function.

OBJECTIVES: Cell transplantation and associated neovascularization in vivo may be beneficial in ischemic disease. We hypothesized that transplanted mesothelial cells (MCs) could improve neovascularization in the post-myocardial infarct scar in rats. METHODS: Myocardial infarction was created by left coronary artery ligation in Lewis rats. After 3 weeks, surviving rats with left ventricular (LV) ejection fraction (EF) <50% were randomized into 2 groups which received, via injection into the infarct scar, either syngeneic rat peritoneal MCs (transplanted group) or vehicle alone (control group). Rats were followed-up echocardiographically for 4 weeks. Before transplantation, cells were transfected in vitro or labeled by a fluorescent dye for subsequent tracking in vivo. Transplanted cells and neovascularization were assessed histologically in the infarct scar by immunostaining or intravenous FITC-dextran injection prior to sacrifice, from 1 to 30 days post-transplantation. RESULTS: Among other pro-angiogenic chemokines, cultured MCs released stromal cell-derived factor (SDF-1alpha) (15.9 +/- 1.8 microg/mg protein) in vitro. At 1 month, some transplanted MCs were visualized (surviving or proliferating) in the LV scar and were incorporated in new vessels. The transplanted rats presented an increased vascular density in the scar, improved LV-EF (44.0 +/- 8.6% vs. 24.0 +/- 4.5%, p < 0.01) with decreased LV end-diastolic diameter (9.6 +/- 0.6 vs. 11.1 +/- 0.6 mm, p < 0.01) and volume (0.47 +/- 0.1 vs. 0.63+/-0.1 ml, p < 0.01) vs. controls. One week post-transplantation, higher levels of SDF-1alpha were extracted from LV peri-infarct tissue (32.3 +/- 5.8 vs. 22.6 +/- 3.1 pg/mg protein in controls, p < 0.01). CONCLUSIONS: Since autologous MCs can be obtained easily and cultured in large quantities, MC transplantation may represent a new angiogenic strategy in the prevention of ischemic remodeling.

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications.

BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.