Giovana Aparecida Gonçalves

Cell Therapy Attenuates Cardiac Dysfunction Post Myocardial Infarction: Effect of Timing, Routes of Injection and a Fibrin Scaffold

Background Cell therapy approaches for biologic cardiac repair hold great promises, although basic fundamental issues remain poorly understood. In the present study we examined the effects of timing and routes of administration of bone marrow cells (BMC) post-myocardial infarction (MI) and the efficacy of an injectable biopolymer scaffold to improve cardiac cell retention and function. Methodology/Principal Findings 99mTc-labeled BMC (6×106 cells) were injected by 4 different routes in adult rats: intravenous (IV), left ventricular cavity (LV), left ventricular cavity with temporal aorta occlusion (LV+) to mimic coronary injection, and intramyocardial (IM). The injections were performed 1, 2, 3, or 7 days post-MI and cell retention was estimated by γ-emission counting of the organs excised 24 hs after cell injection. IM injection improved cell retention and attenuated cardiac dysfunction, whereas IV, LV or LV* routes were somewhat inefficient (<1%). Cardiac BMC retention was not influenced by timing except for the IM injection that showed greater cell retention at 7 (16%) vs. 1, 2 or 3 (average of 7%) days post-MI. Cardiac cell retention was further improved by an injectable fibrin scaffold at day 3 post-MI (17 vs. 7%), even though morphometric and function parameters evaluated 4 weeks later displayed similar improvements. Conclusions/Significance These results show that cells injected post-MI display comparable tissue distribution profile regardless of the route of injection and that there is no time effect for cardiac cell accumulation for injections performed 1 to 3 days post-MI. As expected the IM injection is the most efficient for cardiac cell retention, it can be further improved by co-injection with a fibrin scaffold and it significantly attenuates cardiac dysfunction evaluated 4 weeks post myocardial infarction. These pharmacokinetic data obtained under similar experimental conditions are essential for further development of these novel approaches.

Bone marrow cell therapy prevents infarct expansion and improves border zone remodeling after coronary occlusion in rats

BACKGROUND Since the cell therapy benefits for myocardial infarction are mainly related to infarct reduction by regenerating lost myocardium or increasing survival of tissues at risk, we evaluated the effects of bone marrow-derived mononuclear cells (MNC), implanted after the completion of necrosis, on infarct progression and cardiac remodeling. METHODS After 48 h of induction of myocardial infarction (MI), Lewis-inbred rats were injected with 6 × 10(6) cells (MI+MNC) or saline (MI). After six weeks, scar dimension, ventricular morphology and function were analyzed by echocardiography followed by histomorphology of the infarcted and border zones. RESULTS After therapy, the relative size of the infarct was smaller in MI+MNC (37 ± 1% of the left ventricle) than in MI (43 ± 1%). While the MI group exhibited parallel elongation of the infarcted (31.6 ± 3.8% increase) and reminiscent ventricular portions (33.5 ± 3.7%), MNC therapy preserved the initial infarct length. Infarcted walls were thicker (979 ± 31 mm) in the MNC group than in the untreated group (709 ± 41 mm), also demonstrating an absence of infarct expansion. In the border zones, MNC led to increased capillary densities and capillary/myocyte ratios. The cardiac systolic function remained depressed in MI, but improved by 19 ± 5% in MI+MNC which reduced the incidence of pulmonary arterial hypertension (37.5% in MI and 6.25% in MI+MNC). CONCLUSION MNC therapy prevented the infarct expansion and thinning related to cardiac remodeling and was associated with an improvement of border zone microcirculation: as a result, MNC therapy reduced typical MI dysfunctional repercussions.

Intramyocardial transplantation of fibroblasts expressing vascular endothelial growth factor attenuates cardiac dysfunction

In this study, we analyzed whether transplantation of cardiac fibroblasts (CFs) expressing vascular endothelial growth factor (VEGF) mitigates cardiac dysfunction after myocardial infarction (MI) in rats. First, we observed that the transgene expression lasts longer (45 vs 7 days) when fibroblasts are used as vectors compared with myoblasts. In a preventive protocol, induction of cardiac neovascularization accompanied by reduction in myocardial scar area was observed when cell transplantation was performed 1 week before ischemia/reperfusion and the animals analyzed 3 weeks later. Finally, the therapeutic efficacy of this approach was tested injecting cells in a fibrin biopolymer, to increase cardiac retention, 24 h post-MI. After 4 weeks, an increase in neovascularization and a decrease in myocardial collagen were observed only in rats that received cells expressing VEGF. Basal indirect or direct hemodynamic measurements showed no differences among the groups whereas under pharmacological stress, only the group that received cells expressing VEGF showed a significant reduction in end-diastolic pressure and improvement in stroke volume and cardiac work. These results indicate that transplantation of CFs expressing VEGF using fibrin biopolymer induces neovascularization and attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart.

Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21cip1 in vascular remodelling during vein arterialization in the rat

Neo‐intima development and atherosclerosis limit long‐term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo‐intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while α‐actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin‐dependent kinase inhibitors (CDKIs) p27Kip1 and p16INKA remained unchanged, whereas p21Cip1 was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21Cip1 is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long‐term patency of vein grafts.

Cell therapy prevents structural, functional and molecular remodeling of remote non-infarcted myocardium

BACKGROUND/OBJECTIVES Therapy using bone marrow (BM) cells has been tested experimentally and clinically due to the potential ability to restore cardiac function by regenerating lost myocytes or increasing the survival of tissues at risk after myocardial infarction (MI). In this study we aimed to evaluate whether BM-derived mononuclear cell (MNC) implantation can positively influence the post-MI structural remodeling, contractility and Ca(2+)-handling proteins of the remote non-infarcted tissue in rats. METHODS AND RESULTS After 48 h of MI induction, saline or BM-MNC were injected. Six weeks later, MI scars were slightly smaller and thicker, and cardiac dilatation was just partially prevented by cell therapy. However, the cardiac performance under hemodynamic stress was totally preserved in the BM-MNC treated group if compared to the untreated group, associated with normal contractility of remote myocardium as analyzed in vitro. The impaired post-rest potentiation of contractile force, associated with decreased protein expression of the sarcoplasmic reticulum Ca(2+)-ATPase and phosphorylated-phospholamban and overexpression of Na(+)/Ca(2+) exchanger, were prevented by BM-MNC, indicating preservation of the Ca(2+) handling. Finally, pathological changes on remodeled remote tissue such as myocyte hypertrophy, interstitial fibrosis and capillary rarefaction were also mitigated by cell therapy. CONCLUSIONS BM-MNC therapy was able to prevent cardiac structural and molecular remodeling after MI, avoiding pathological changes on Ca(2+)-handling proteins and preserving contractile behavior of the viable myocardium, which could be the major contributor to the improvements of global cardiac performance after cell transplantation despite that scar tissue still exists.

17β-Estradiol and steady-state concentrations of H2O2: antiapoptotic effect in endometrial cells from patients with endometriosis

Increased levels of hydrogen peroxide (H2O2) can initiate protective responses to limit or repair oxidative damage. However, H2O2 signals also fine-tune responses to growth factors and cytokines controlling cell division, differentiation, and proliferation. Because 17β-estradiol (E2) also plays important roles in these processes, and is considered a major risk factor in the development and progression of endometriosis, this study evaluated whether E2 has an antiapoptotic effect on oxidative stress in endometrial cells in combination with steady-state H2O2 levels ([H2O2]ss). Endometrial stromal cells were prepared from the eutopic endometrium of 18 women with and without endometriosis to produce primary cells. These cells were stimulated with E2 for 20h, exposed to [H2O2]ss, and examined for cell viability, proliferation, and apoptosis. The endometrial cells from women with endometriosis maintained the steady state for 120min at high H2O2 concentrations. When they were pretreated with E2 and exposed to [H2O2]ss, a decrease in apoptosis level was observed compared to the control cells (p<0.01). The endometrial cells from patients with endometriosis subjected to both E2 and [H2O2]ss showed increased ERK phosphorylation. These findings suggested that H2O2 is a signaling molecule that downregulates apoptosis in endometrial cells, supporting the fact that endometriosis, albeit a benign disease, shares some features with cancer such as decreased catalase levels. These results link the E2 effects on [H2O2]ss to resistance to apoptosis and progression of endometriosis.

KIR and a specific HLA-C gene are associated with susceptibility and resistance to hepatitis B virus infection in a Brazilian population

KIR and a specific HLA-C gene are associated with susceptibility and resistance to hepatitis B virus infection in a Brazilian population

p27kip1 overexpression regulates IL-1β in the microenvironment of stem cells and eutopic endometriosis co-cultures

Highlightsp27kip1 gene therapy can decrease the IL‐1&bgr; production in endometriosis.p27kip1 gene therapy as an alternative strategy in the treatment of endometriosis.p27kip1 gene therapy as an regulator of Il‐1&bgr; in endometriosis. ABSTRACT Endometriosis is a gynecological benign chronic disease defined as the growth of endometrial glands and stroma in extra‐uterine sites, most commonly implanted over visceral and peritoneal surfaces within the female pelvis causing inflammatory lesions. It affects around 10% of the female population and is often accompanied by chronic pelvic pain, adhesion formation and infertility. Therefore, endometriosis could be considered a “social disease”, since it affects the quality of life, reproductivity and also has a socio‐economic impact. The expression of cell cycle and inflammatory proteins is modified in the endometriotic tissues. Immunostaining of glandular and stromal cells in endometrial biopsies obtained from patients with endometriosis compared with those of healthy control demonstrated that endometriotic tissues have lower levels of p27kip1 protein. Endometriosis endometrial cells cultures have also lower levels of p27kip1 compared to health endometrial cells cultures and restore the cell cycle balance when transduced with an adenoviral vector carring the p27kip1 coding gene (Adp27EGFP). The low levels of p27kip1 are related to the S phase in the cell cycle, whereas higher levels lead to a G1 cell cycle arrest. The inflammatory cytokine IL‐1&bgr; was recently identified as another key protein in the endometriosis proliferation. This cytokine has elevated levels during the proliferative and secretory phases of the menstrual cycle. In endometriosis endometrial cells cultures the IL‐1&bgr; stimulates the production of IL‐6 and IL‐8, increasing the cell proliferation and reducing the apoptosis and Bax expression in these cells. According to these remarks, this work aims to evaluate the inflammatory effects in vitro, but more next to what happens in a womans body, associating endometrial cells with stem cells, thus mimicking the endometrial microenvironment, with gene therapy using Adp27, notoriously known as controller cell cycle, apoptosis and potent modulator of VEGF expression.

Inflammatory cytokine profile of co‑cultivated primary cells from the endometrium of women with and without endometriosis

Endometriosis is a chronic gynecological disorder defined as the presence of endometrial tissue within extra-uterine sites. The primary symptoms are infertility and chronic pain. The inflammatory environment and aberrant immune responses in women with endometriosis may be directly associated with the initiation and progression of endometriotic lesions. In the present study, the secretion of inflammatory cytokines was evaluated in cultures of primary endometrial cells (ECs) isolated from the endometrium of women with and without endometriosis. The presence of endometriotic cells leads to alterations in the secretory profile of healthy ECs. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was significantly increased in endometriotic and co-cultured cells compared with healthy ECs. IL-6 expression was strongly correlated with IL-8 expression in endometriotic cells. IL-1β expression was increased on day 10 of co-culture to 48.30 pg/ml and may be associated with the long-term co-culture, rather than IL-6 and IL-8 expression. IL-6 expression was strongly correlated with cell number, whereas IL-8 expression was moderately correlated with cell number. Additionally, it was observed that co-cultured cells exhibited a different population of cells, with expression of the mesenchymal stem cell marker cell surface glycoprotein MUC18, indicating a putative role of endometrial mesenchymal stem cells in the secretion of cytokines and disease development. These results indicate a predominant role of primary endometriotic cells in the secretion of cytokines, which contributes to the disrupted peritoneal and endometrial environment observed in the women with endometriosis.