The Role of Plasma Extracellular Vesicles in Remote Ischemic Conditioning and Exercise-Induced Ischemic Tolerance

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.     

Citric Acid Cycle Metabolites Predict Infarct Size in Pigs Submitted to Transient Coronary Artery Occlusion and Treated with Succinate Dehydrogenase Inhibitors or Remote Ischemic Perconditioning

Succinate dehydrogenase (SDH) inhibition with malonate during reperfusion reduced myocardial infarction in animals, whereas its endogenous substrate, succinate, is detected in plasma from STEMI patients. We investigated whether protection by SDH inhibition is additive to that of remote ischemic perconditioning (RIC) in pigs submitted to transient coronary artery occlusion, and whether protective maneuvers influence plasma levels of citric acid cycle metabolites. Forty pigs were submitted to 40 min coronary occlusion and reperfusion, and allocated to four groups (controls, sodium malonate 10 mmol/L, RIC, and malonate + RIC). Plasma was obtained from femoral and great cardiac veins and analyzed by LC-MS/MS. Malonate, RIC, and malonate + RIC reduced infarct size (24.67 ± 5.98, 25.29 ± 3.92 and 29.83 ± 4.62% vs. 46.47 ± 4.49% in controls, p < 0.05), but no additive effects were detected. Enhanced concentrations of succinate, fumarate, malate and citrate were observed in controls during initial reperfusion in the great cardiac vein, and most were reduced by cardioprotective maneuvers. Concentrations of succinate, fumarate, and malate significantly correlated with infarct size. In conclusion, despite the combination of SDH inhibition during reperfusion and RIC did not result in additive protection, plasma concentrations of selected citric acid cycle metabolites are attenuated by protective maneuvers, correlate with irreversible injury, and might become a prognosis tool in STEMI patients.     

Ischemic Postconditioning Alleviates Neuronal Injury Caused by Relief of Carotid Stenosis in a Rat Model of Cerebral Hypoperfusion

The effects of early relief of heavy bilateral carotid stenosis and ischemic postconditioning on hippocampus CA1 neurons are still unclear. In this study, we used a rat model to imitate severe bilateral carotid stenosis in humans. The rats were divided into sham group, carotid stenosis group, stenosis relief group and ischemic postconditioning group. Ischemic postconditioning consisted of three cycles of 30 s ischemia and 30 s reperfusion. The cerebral blood flow was measured with a laser Doppler flowmeter. Neuronal death in the CA1 region was observed by hematoxylin-eosin staining, and the number of live neurons was assessed by cell counting under a light microscope. The levels of oxidative products MDA and 8-iso-PGF2α, inflammatory factors IL-1β and TNF-α, and the activities of anti-oxidative enzymes SOD and CAT were assayed by specific enzyme-linked immunosorbent assay (ELISA) kits, respectively. We found that relief of carotid stenosis and ischemic postconditioning could increase cerebral blood flow. When stenosis was relieved, the percentage of live neurons was 66.6% ± 6.2% on day 3 and 62.3% ± 9.8% on day 27, which was significantly higher than 55.5% ± 4.8% in stenosis group. Ischemic postconditioning markedly improved the live neurons to 92.5% ± 6.7% on day 3 and 88.6% ± 9.1% on day 27. Further study showed that, neuronal death caused by relief of stenosis is associated with increased oxidative stress and enhanced inflammatory response, and the protection of ischemic postconditioning is related to inhibition of oxidative stress and suppression of inflammatory response.     

Comparison of Repeated Doses of C-kit-Positive Cardiac Cells versus a Single Equivalent Combined Dose in a Murine Model of Chronic Ischemic Cardiomyopathy

Using a murine model of chronic ischemic cardiomyopathy caused by an old myocardial infarction (MI), we have previously found that three doses of 1 × 10⁶ c-kit positive cardiac cells (CPCs) are more effective than a single dose of 1 × 10⁶ cells. The goal of this study was to determine whether the beneficial effects of three doses of CPCs (1 × 10⁶ cells each) can be fully replicated by a single combined dose of 3 × 10⁶ CPCs. Mice underwent a 60-min coronary occlusion; after 90 days of reperfusion, they received three echo-guided intraventricular infusions at 5-week intervals: (1) vehicle × 3; (2) one combined dose of CPCs (3 × 10⁶) and vehicle × 2; or (3) three doses of CPCs (1 × 10⁶ each). In the combined-dose group, left ventricular ejection fraction (LVEF) improved after the 1st CPC infusion, but not after the 2nd and 3rd (vehicle) infusions. In contrast, in the multiple-dose group, LVEF increased after each CPC infusion; at the final echo, LVEF averaged 35.2 ± 0.6% (p < 0.001 vs. the vehicle group, 27.3 ± 0.2%). At the end of the study, the total cumulative change in EF from pretreatment values was numerically greater in the multiple-dose group (6.6 ± 0.6%) than in the combined-dose group (4.8 ± 0.8%), although the difference was not statistically significant (p = 0.08). Hemodynamic studies showed that several parameters of LV function in the multiple-dose group were numerically greater than in the combined-dose group (p = 0.08 for the difference in LVEF). Compared with vehicle, cardiomyocyte cross-sectional area was reduced only in the multiple-dose group (−32.7%, 182.6 ± 15.1 µm² vs. 271.5 ± 27.2 µm², p < 0.05, in the risk region and −28.5%, 148.5 ± 12.1 µm² vs. 207.6 ± 20.5 µm², p < 0.05, in the noninfarcted region). LV weight/body weight ratio and LV weight/tibia length ratios were significantly reduced in both cell treated groups vs. the vehicle group, indicating the attenuation of LV hypertrophy; however, the lung weight/body weight ratio was significantly reduced only in the multiple-dose group, suggesting decreased pulmonary congestion. Taken together, these results indicate that in mice with chronic ischemic cardiomyopathy, the beneficial effects of three doses of CPCs on LV function and hypertrophy cannot be fully replicated with a single dose, notwithstanding the fact that the total number of cells delivered with one or three doses is the same. Thus, it is the multiplicity of doses, and not the total number of cells, that accounts for the superiority of the repeated-dose paradigm. This study supports the idea that the efficacy of cell therapy in heart failure can be augmented by repeated administrations.     

Time-Dependent Changes in Hepatic Sphingolipid Accumulation and PI3K/Akt/mTOR Signaling Pathway in a Rat Model of NAFLD

Increased lipid bioavailability in a diet favors lipid accumulation, enhancing hepatic lipotoxicity and contributing to insulin resistance (IR) development. The aim of our study was to examine time-dependent alterations in the intrahepatic content of sphingolipids and insulin signaling pathway in rats fed a high-fat diet (HFD). The experiment was conducted on male Wistar rats receiving a standard diet or HFD for five weeks. At the end of each experimental feeding week, liver sphingolipids were determined using high-performance liquid chromatography. The expression of proteins from the sphingolipid pathway and glucose transporter expression were assessed by Western blot. The content of phosphorylated form of proteins from the insulin pathway was detected by a multiplex assay kit. Our results revealed that HFD enhanced hepatic ceramide deposition by increasing the expression of selected proteins from sphingomyelin and salvage pathways in the last two weeks. Importantly, we observed a significant inhibition of Akt phosphorylation in the first week of HFD and stimulation of PTEN and mTOR phosphorylation at the end of HFD. These changes worsened the PI3K/Akt/mTOR signaling pathway. We may postulate that HFD-induced reduction in the insulin action in the time-dependent matter was exerted by excessive accumulation of sphingosine and sphinganine rather than ceramide.     

Quercetin Improves Postischemic Recovery of Heart Function in Doxorubicin-Treated Rats and Prevents Doxorubicin-Induced Matrix Metalloproteinase-2 Activation and Apoptosis Induction

Quercetin (QCT) is flavonoid that possesses various biological functions including anti-oxidative and radical-scavenging activities. Moreover, QCT exerts some preventive actions in treatment of cardiovascular diseases. The aim of present study was to explore effects of prolonged administration of QCT on changes induced by repeated application of doxorubicin (DOX) in rat hearts. We focused on the ultrastructure of myocardium, matrix metalloproteinases (MMPs), biometric parameters, and apoptosis induction. Our aim was also to examine effects of QCT on ischemic tolerance in hearts exposed to chronic effects of DOX, and to determine possible mechanisms underlying effects of QCT. Our results showed that QCT prevented several negative chronic effects of DOX: (I) reversed DOX-induced blood pressure increase; (II) mediated improvement of deleterious effects of DOX on ultrastructure of left ventricle; (III) prevented DOX-induced effects on tissue MMP-2 activation; and (iv) reversed effects of DOX on apoptosis induction and superoxide dismutase inhibition. Moreover, we showed that rat hearts exposed to effects of QCT were more resistant to ischemia/reperfusion injury. Effects of QCT on modulation of ischemic tolerance were linked to Akt kinase activation and connexin-43 up-regulation. Taken together, these results demonstrate that prolonged treatment with QCT prevented negative chronic effects of DOX on blood pressure, cellular damage, MMP-2 activation, and apoptosis induction. Moreover, QCT influenced myocardial responses to acute ischemic stress. These facts bring new insights into mechanisms of QCT action on rat hearts exposed to the chronic effects of DOX.     

Intermittent Hypoxia on the Attenuation of Induced Nasal Allergy and Allergic Asthma by MAPK Signaling Pathway Downregulation in a Mice Animal Model

Intermittent hypoxia (IH) has been an issue of considerable research in recent years and triggers a bewildering array of both detrimental and beneficial effects in several physiological systems. However, the mechanisms leading to the effect are not yet clear. Consequently, we investigated the effects of IH on allergen-induced allergic asthma via the mitogen-activated protein kinase (MAPK) signaling pathway. Forty BALB/c mice were dived into four groups. We evaluated the influence of IH on the cell signaling system of the airway during the allergen-induced challenge in an animal model, especially through the MAPK (mitogen-activated protein kinase) pathway. The protein concentrations of p-ERK/ERK, p-JNK/JNK, p-p38/p38, and pMEK/MEK were significantly reduced in the allergen-induced+IH group, compared to the allergen-induced group (p-value < 0.05 as considered statistically significant). The number of eosinophils, neutrophils, macrophages, and lymphocytes in the bronchoalveolar lavage fluid and Dp (Dermatophagoides pteronyssinus)-specific IgG2a and interleukins 4, 5, 13, and 17 were significantly reduced in the Dp+IH group, compared to the Dp group. These findings suggest that the MAPK pathway might be associated with the beneficial effect of IH on the attenuation of allergic response in an allergen-induced mouse model.     

Antiviral and Anti-Inflammatory Activities of Fluoxetine in a SARS-CoV-2 Infection Mouse Model

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world’s population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.     

FAK-Mediated Signaling Controls Amyloid Beta Overload,Learning and Memory Deficits in a Mouse Model of Alzheimer’s Disease

The non-receptor focal adhesion kinase (FAK) is highly expressed in the central nervous system during development, where it regulates neurite outgrowth and axon guidance, but its role in the adult healthy and diseased brain, specifically in Alzheimer’s disease (AD), is largely unknown. Using the 3xTg-AD mouse model, which carries three mutations associated with familial Alzheimer’s disease (APP KM670/671NL Swedish, PSEN1 M146V, MAPT P301L) and develops age-related progressive neuropathology including amyloid plaques and Tau tangles, we describe here, for the first time, the in vivo role of FAK in AD pathology. Our data demonstrate that while site-specific knockdown in the hippocampi of 3xTg-AD mice has no effect on learning and memory, hippocampal overexpression of the protein leads to a significant decrease in learning and memory capabilities, which is accompanied by a significant increase in amyloid β (Aβ) load. Furthermore, neuronal morphology is altered following hippocampal overexpression of FAK in these mice. High-throughput proteomics analysis of total and phosphorylated proteins in the hippocampi of FAK overexpressing mice indicates that FAK controls AD-like phenotypes by inhibiting cytoskeletal remodeling in neurons which results in morphological changes, by increasing Tau hyperphosphorylation, and by blocking astrocyte differentiation. FAK activates cell cycle re-entry and consequent cell death while downregulating insulin signaling, thereby increasing insulin resistance and leading to oxidative stress. Our data provide an overview of the signaling networks by which FAK regulates AD pathology and identify FAK as a novel therapeutic target for treating AD.     

Carbamazepine Restores Neuronal Signaling,Protein Synthesis,and Cognitive Function in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is a leading genetic disorder of intellectual disability caused by the loss of the functional fragile X mental retardation protein (FMRP). To date, there is no efficacious mechanism-based medication for FXS. With regard to potential disease mechanisms in FXS, it is widely accepted that the lack of FMRP causes elevated protein synthesis and deregulation of neuronal signaling. Abnormal enhancement of the ERK½ (extracellular signal-regulated kinase ½) and PI3K-Akt (Phosphoinositide 3 kinase-protein kinase B) signaling pathways has been identified in both FXS patients and FXS mouse models. In this study, we show that carbamazepine, which is an FDA-approved drug and has been mainly used to treat seizure and neuropathic pain, corrects cognitive deficits including passive avoidance and object location memory in FXS mice. Carbamazepine also rescues hyper locomotion and social deficits. At the cellular level, carbamazepine dampens the elevated level of ERK½ and Akt signaling as well as protein synthesis in FXS mouse neurons. Together, these results advocate repurposing carbamazepine for FXS treatment.     

Simvastatin Attenuates the Oxidative Stress,Endothelial Thrombogenicity and the Inducibility of Atrial Fibrillation in a Rat Model of Ischemic Heart Failure

Increased atrial oxidative stress has an important role in inducing and maintaining atrial fibrillation (AF), and the activation of the small GTPase Rac1 contributes to the oxidative stress. We investigated the relationship of Rac1, atrial endothelial thromboprotective markers and AF inducibility and if simvastatin has a potential beneficial effect on a myocardial infarction (MI)-induced heart failure (HF) rat model. Rats were randomized into three groups (shams, MI group and simvastatin treatment group) and underwent echocardiography, AF induction studies and left atrial (LA) fibrosis analysis. Atrial Rac 1, sodium calcium exchanger (I_NCX), sarcoplasmic reticulum calcium ATPase (SERCA), endothelial nitric oxide synthase (eNOS) and induced nitric oxide synthase (iNOS) were measured. AF inducibility, AF duration and LA fibrosis were significantly higher in the MI group (p < 0.001 vs. sham), which were significantly reduced by simvastatin (p < 0.05 vs. MI). The reduced expressions of atrial eNOS, SERCA, thrombomodulin, tissue factor pathway inhibitor and tissue plasminogen activator in the MI group were significantly improved by simvastatin. Furthermore, the increased expression of atrial iNOS, I_NCX and Rac1 activity were significantly decreased by the simvastatin. Oxidative stress, endothelial dysfunction and thrombogenicity are associated with the promotion of AF in a rat model of ischemic HF. These were associated with increased Rac1 activity, and simvastatin treatment prevents these changes.     

Puerarin-V Improve Mitochondrial Respiration and Cardiac Function in a Rat Model of Diabetic Cardiomyopathy via Inhibiting Pyroptosis Pathway through P2X7 Receptors

There is a new form of puerarin, puerarin-V, that has recently been developed, and it is unclear whether puerarin-V has a cardioprotective effect on diabetic cardiomyopathy (DCM). Here, we determined whether puerarin-V had any beneficial influence on the pathophysiology of DCM and explored its possible mechanisms. By injecting 30 mg/kg of STZ intraperitoneally, diabetes was induced in rats. After a week of stability, the rats were injected subcutaneously with ISO (5 mg/kg). We randomly assigned the rats to eight groups: (1) control; (2) model; (3) metformin; (4–6) puerarin-V at different doses; (7) puerarin (API); (8) puerarin injection. DCM rats were found to have severe cardiac insufficiency (arrythmia, decreased LVdP/dt, and increased E/A ratio). In addition, cardiac injury biomarkers (cTn-T, NT-proBNP, AST, LDH, and CK-MB), inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α), and oxidative damage markers (MDA, SOD and GSH) were markedly increased. Treatment with puerarin-V positively adjusts these parameters mentioned above by improving cardiac function and mitochondrial respiration, suppressing myocardial inflammation, and maintaining the structural integrity of the cardiac muscle. Moreover, treatment with puerarin-V inhibits the P2X7 receptor-mediated pyroptosis pathway that was upregulated in diabetic hearts. Given these results, the current study lends credence to the idea that puerarin-V can reduce myocardial damage in DCM rats. Furthermore, it was found that the effect of puerarin-V in diabetic cardiomyopathy is better than the API, the puerarin injection, and metformin. Collectively, our research provides a new therapeutic option for the treatment of DCM in clinic.     

Investigation of the Antihypertrophic and Antifibrotic Effects of Losartan in a Rat Model of Radiation-Induced Heart Disease

Radiation-induced heart disease (RIHD) is a potential late side-effect of thoracic radiotherapy resulting in left ventricular hypertrophy (LVH) and fibrosis due to a complex pathomechanism leading to heart failure. Angiotensin-II receptor blockers (ARBs), including losartan, are frequently used to control heart failure of various etiologies. Preclinical evidence is lacking on the anti-remodeling effects of ARBs in RIHD, while the results of clinical studies are controversial. We aimed at investigating the effects of losartan in a rat model of RIHD. Male Sprague-Dawley rats were studied in three groups: (1) control, (2) radiotherapy (RT) only, (3) RT treated with losartan (per os 10 mg/kg/day), and were followed for 1, 3, or 15 weeks. At 15 weeks post-irradiation, losartan alleviated the echocardiographic and histological signs of LVH and fibrosis and reduced the overexpression of chymase, connective tissue growth factor, and transforming growth factor-beta in the myocardium measured by qPCR; likewise, the level of the SMAD2/3 protein determined by Western blot decreased. In both RT groups, the pro-survival phospho-AKT/AKT and the phospho-ERK1,2/ERK1,2 ratios were increased at week 15. The antiremodeling effects of losartan seem to be associated with the repression of chymase and several elements of the TGF-β/SMAD signaling pathway in our RIHD model.     

Effects and Mechanism of Particulate Matter on Tendon Healing Based on Integrated Analysis of DNA Methylation and RNA Sequencing Data in a Rat Model

Exposure to particulate matter (PM) has been linked with the severity of various diseases. To date, there is no study on the relationship between PM exposure and tendon healing. Open Achilles tenotomy of 20 rats was performed. The animals were divided into two groups according to exposure to PM: a PM group and a non-PM group. After 6 weeks of PM exposure, the harvest and investigations of lungs, blood samples, and Achilles tendons were performed. Compared to the non-PM group, the white blood cell count and tumor necrosis factor-alpha expression in the PM group were significantly higher. The Achilles tendons in PM group showed significantly increased inflammatory outcomes. A TEM analysis showed reduced collagen fibrils in the PM group. A biomechanical analysis demonstrated that the load to failure value was lower in the PM group. An upregulation of the gene encoding cyclic AMP response element-binding protein (CREB) was detected in the PM group by an integrated analysis of DNA methylation and RNA sequencing data, as confirmed via a Western blot analysis showing significantly elevated levels of phosphorylated CREB. In summary, PM exposure caused a deleterious effect on tendon healing. The molecular data indicate that the action mechanism of PM may be associated with upregulated CREB signaling.     

Protective Effects of Currants (Vitis vinifera) on Corticolimbic Serotoninergic Alterations and Anxiety-like Comorbidity in a Rat Model of Parkinson’s Disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of nigral dopaminergic neurons. Increasing evidence supports that PD is not simply a motor disorder but a systemic disease leading to motor and non-motor symptoms, including memory loss and neuropsychiatric conditions, with poor management of the non-motor deficits by the existing dopaminergic medication. Oxidative stress is considered a contributing factor for nigrostriatal degeneration, while antioxidant/anti-inflammatory properties of natural phyto-polyphenols have been suggested to have beneficial effects. The present study aimed to determine the contribution of monoaminergic neurotransmission on the anxiety-like phenotype in a rat rotenone PD model and evaluate the possible neuroprotective effects of black Corinthian currant, Vitis vinifera, consisting of antioxidant polyphenols. Rotenone-treated rats showed anxiety-like behavior and exploratory deficits, accompanied by changes in 5-HT, SERT and β₂-ARs expression in the prefrontal cortices, hippocampus and basolateral amygdala. Importantly, the motor and non-motor behavior, as well as 5-HT, SERT and β₂-ARs expression patterns of the PD-like phenotype were partially recovered by a supplementary diet with currants. Overall, our results suggest that the neuroprotective effects of Corinthian currants in rotenone-induced anxiety-like behavior may be mediated via corticolimbic serotonergic transmission.     

Effect of Ion Concentration Changes in the Limited Extracellular Spaces on Sarcolemmal Ion Transport and Ca2+ Turnover in a Model of Human Ventricular Cardiomyocyte

We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca²⁺]_i-transient was explored as a function of critical fractions of ion transporters in t-tubular membrane (not documented for human CVM). Depletion of Ca²⁺ and accumulation of K⁺ occurring in extracellular spaces slightly affected the transmembrane Ca²⁺ flux, but not the action potential duration (APD₉₀). The [Ca²⁺]_i-transient was reduced (by 2%–9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1–2.5 Hz) caused a temporal decrease of [Ca²⁺] in both extracellular spaces, a reduction of [Ca²⁺]_i-transient (by 15%) and APD₉₀ (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca²⁺ turnover.     

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated p16 expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.     

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24–28%; 0.17–0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.