Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice.

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund's adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Vasohibin-1 rescues erectile function through up-regulation of angiogenic factors in the diabetic mice.

Neovascularization of the erectile tissue emerges as a beneficial curative approach to treat erectile dysfunction (ED). Here we for the first time report the unexpected role of vasohibin-1 (VASH1), mainly known as an anti-angiogenic factor, in restoring erectile function in diabetic mice. A diabetic patient has lower cavernous VASH1 expression than in the potent man. VASH1 was mainly expressed in endothelial cells. There were significant decreases in cavernous endothelial cell and pericyte contents in VASH1 knockout mice compared with those in wild-type mice, which resulted in impairments in erectile function. Intracavernous injection of VASH1 protein successfully restored erectile function in the diabetic mice (~ 90% of control values). VASH1 protein reinstated endothelial cells, pericytes, and endothelial cell-cell junction proteins and induced phosphorylation of eNOS (Ser1177) in the diabetic mice. The induction of angiogenic factors, such as angiopoietin-1 and vascular endothelial growth factor, is responsible for cavernous angiogenesis and the restoration of erectile function mediated by VASH1. Altogether, these findings suggest that VASH1 is proangiogenic in diabetic penis and is a new potential target for diabetic ED.

Co-delivery of EGFR and BRD4 siRNA by cell-penetrating peptides-modified redox-responsive complex in triple negative breast cancer cells.

AIMS: Triple negative breast cancer (TNBC) has drawn more and more attention due to its high mitotic indices, high metastatic rate and poor prognosis. Gene therapy, especially RNA interference (RNAi), has become a promising targeted therapy. However, improvement of transfection efficiency and discovery of target genes are major problems for the delivery of small interfering RNAs (siRNA). MATERIALS AND METHODS: In the present study, we developed GALA- and CREKA-modified PEG-SS-PEI to deliver siRNAs targeting on EGFR and BRD4 for TNBC therapy. The PEG-SS-PEI/siRNA complexes were prepared by electrostatic interaction and characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). The release characteristic, stability, cellular uptake and intracellular localization of the complexes were also studied. The effect of the complexes on cell viability was measured in MDA-MB-231 and HUVEC cells. The in vitro anti-tumor activities of the complexes were analyzed by Transwell invasion assay and wound healing assay. The gene silencing effect was evaluated by quantitative real time-polymerase chain reaction (qRT-PCR) and western blot. KEY FINDINGS: The results revealed that the GALA- and CREKA-modified PEG-SS-PEI/siRNA complexes showed excellent transfection efficiency with redox-sensitive release profile and good biological compatibility. The complexes protected siRNA from the degradation of RNA enzymes. The complexes significantly inhibited the proliferation, invasion and migration of MDA-MB-231 cells via the synergistic inhibition of EGFR/PI3K/Akt and BRD4/c-Myc pathways. SIGNIFICANCE: Taken together, co-delivery of siEGFR and siBRD4 by GALA-PEG-SS-PEI and CREKA-PEG-SS-PEI may provide a more effective strategy for the treatment of TNBC.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

mInscuteable regulates meiotic spindle organization during mouse oocyte meiotic maturation.

Establishment of cellular polarity is one of the key events during oocyte maturation. Inscuteable (Insc) has been identified as a key regulator of cell polarity during asymmetric division in Drosophila. However, the function of its evolutionarily conserved mammalian homologue, mInscuteable (mInsc), in mouse meiotic maturation is not clear. In this study, we investigated the roles of mInsc in mouse oocyte maturation. mInsc was detected at all stages of oocyte maturation. The protein level of mInsc was slightly higher at the germinal vesicle breakdown (GVBD) stage and remained constant during mouse oocyte maturation. The subcellular localization of mInsc overlapped with spindle microtubules. Disruption of microtubules and microfilaments caused changes in the localization of mInsc. Depletion or overexpression of mInsc significantly decreased the maturation rates of mouse oocytes. Depletion of mInsc significantly affected asymmetric division, spindle assembly, alignments of chromosomes and actin cap formation. Taken together, our results demonstrated that mInsc regulates meiotic spindle organization during mouse meiotic maturation.

Structural basis for Scc3-dependent cohesin recruitment to chromatin.

The cohesin ring complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair and transcriptional regulation. How cohesin engages its chromatin substrate has remained an unresolved question. We show here, by determining a crystal structure of the budding yeast cohesin HEAT-repeat subunit Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.

Additive effects of the combined expression of soluble forms of GAS1 and PTEN inhibiting glioblastoma growth.

The overexpression of GAS1 (Growth Arrest Specific 1) in glioma cells induces cell cycle arrest and apoptosis. We previously demonstrated that the apoptotic process set off by GAS1 is caused by its capacity to inhibit the Glial cell-derived neurotrophic factor (GDNF)-mediated intracellular survival signaling pathway. Whereas on the other hand, PTEN is a tumor suppressor, inactive in many tumors, and both GAS1 and PTEN inhibit the PI3K/AKT pathway. Therefore, it is relevant to investigate the potential additive effect of the overexpression of GAS1 and PTEN on tumor growth. In particular, we employed secreted forms of both GAS1 (tGAS1) and PTEN (PTEN-LONG, or PTEN-L) and tested their combined effect on glioma cells. We observed that the co-expression of both the proteins inhibited the growth of U-87 MG human glioblastoma cells more effectively than when independently expressed, and decreased the activity of both AKT and ERK1/2. Interestingly, the combination of the soluble forms was always the most effective treatment. To improve the transfer of tGAS1 and PTEN-L, we employed a lentiviral vector with a p2A peptide-enabled dual expression system that allowed the generation of the two proteins using a single promoter (CMV), in equimolar amounts. The viral vector reduced the growth of U-87 MG cells in vitro and had a striking effect in inhibiting their proliferation after inoculating it into the immunosuppressed mice. The present results support a potential adjuvant role for the combined use of tGAS1 and PTEN-L in the treatment of glioblastoma.

The enhancement of siPLK1 penetration across BBB and its anti glioblastoma activity in vivo by magnet and transferrin co-modified nanoparticle.

In order to enhance the penetration of small interference RNA against the polo-like kinase I (siPLK1) across BBB to treat glioblastoma (GBM), transferrin (Tf) modified magnetic nanoparticle (Tf-PEG-PLL/MNP@siPLK1) was prepared. The in vitro experiments indicated that Tf-PEG-PLL/MNP@siPLK1 enhanced the cellular uptake of siPLK1, which resulted in an increase of gene silencing effect and cytotoxicity of Tf-PEG-PLL/MNP@siPLK1 on U87 cells. Besides, Tf-PEG-PLL/MNP@siPLK1 significantly inhibited the growth of U87 glioblastoma spheroids and markedly increased the BBB penetration efficiency of siPLK1 with the application of external magnetic field in in-vitro BBB model. The in vivo experiments indicated that siPLK1 selectively accumulated in the brain tissue, and markedly reduced tumor volume and prolonged the survival time of GBM-bearing mice after Tf-PEG-PLL/MNP@siPLK1 was injected to GBM-bearing mice via tail vein. The above data indicated that magnet and transferrin co-modified nanoparticle enhanced siPLK1 penetration across BBB and increased its anti GBM activity in vivo.

Carbon nanospheres mediated nuclear delivery of SMAR1 protein (DNA binding domain) controls breast tumor in mice model.

AIM: To investigate anticancer activity of the DNA binding domain of SMAR1 (His 5) in vitro and in vivo. MATERIALS & METHODS: His 5 was conjugated to hydrothermally synthesized carbon nanospheres (CNs). Anticancer activity of CNs-His 5 was evaluated in vitro and in vivo. RESULTS: CNs- His 5 significantly reduced cyclin D1 levels in MDA-MB-231 cells. Tumor bearing Balb/c mice injected with CNs-His 5 showed approximately 62% tumor regression and significantly reduced 18FDG uptake. Caspases assay and IHC staining confirmed tumor growth inhibition, which could be attributed to apoptotic, antiproliferative and antiangiogenic activities of His 5. CONCLUSION: DNA binding domain of the SMAR1 protein (His 5) has potent anticancer activity and its CNs mediated delivery could control breast tumor in mice model.

Role of inhibitor of growth 4 in the suppression of human melanoma cells through the Fas/FasL-mediated apoptosis pathway.

Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current available therapies. Inhibitor of growth 4 (ING4), a novel member of the ING family of proteins, has previously been shown to play a critical role in the development of multiple tumors by regulating apoptosis, proliferation, cell cycle progress, migration and invasion. However, the functional role of ING4 in human melanoma remains unclear. To fully understand its potential role in human melanoma, in the present study, lentivirus (LV)­ING4 and LV­ING4­short hairpin RNA were constructed and transfected into human melanoma A375 cells. First, the effect of overexpressing or downregulating ING4 on the apoptosis of the transfected melanoma cells and cluster of differentiation (CD)3+ T cells was investigated. In the present study, we found that the late apoptotic cells, and not the early apoptotic cells, were more in LV-ING4 group compared with LV-control, and both the early and late apoptosis of CD3+ T cells was significantly observed in A375 cells transfected with LV-ING4 compared with LV-control. Importantly, it was determined whether the overexpression of ING4 significantly induce apoptotic cell death via Fas/FasL (Fas death receptor/FasL) pathway activation and downregulation of poly(ADP­ribose) polymerase, caspase­3 and caspase­8 in the melanoma cells and CD3+ T cells. These results demonstrated that overexpression of ING4 can induce the apoptosis of melanoma cells and CD3+ T cells through signaling pathways such as the Fas/FasL pathway, and that ING4 gene therapy for melanoma treatment is a novel approach.

Co-delivery of doxorubicin and recombinant plasmid pHSP70-Plk1-shRNA by bacterial magnetosomes for osteosarcoma therapy.

To explore a novel combination of chemotherapy, gene therapy, and thermotherapy for osteosarcoma, a targeted heat-sensitive co-delivery system based on bacterial magnetosomes (BMs) was developed. The optimal culture conditions of magnetotactic bacteria (MTB) AMB-1 and characterization of BMs were achieved. A recombinant eukaryotic plasmid heat shock protein 70-polo-like kinase 1-short hairpin RNA (pHSP70-Plk1-shRNA) under transcriptional control of a thermosensitive promoter (human HSP70 promoter) was constructed for gene therapy. Doxorubicin (DOX) and pHSP70-Plk1-shRNA were included in the targeted thermosensitive co-delivery system, and in vitro DOX release activity, targeted gene silencing efficiency and in vitro antitumor efficacy were investigated. The results showed that the optimal culture conditions of MTB AMB-1 are an oxygen concentration of 4.0%, a pH value of 7.0, 20 µmol/L of ferrous sulfate, 800 mg/L of sodium nitrate, and 200 mg/L of succinic acid. The temperature of BMs reached 43°C within 3 minutes and could be maintained for 30 minutes by adjusting the magnitude of the alternating magnetic field (AMF). The diameters of BMs, BM-DOX, BM-recombinant eukaryotic plasmid pHSP70-Plk1-shRNA (shPlk1), and BM-DOX-shPlk1 were 43.7±4.6, 79.2±5.4, 88.9±7.8, and 133.5±11.4 nm, respectively. The zeta potentials of BMs, BM-DOX, BM-shPlk1, and BM-DOX-shPlk1 were -29.4±6.9, -9.5±5.6, -16.7±4.8, and -10.3±3.1 mV, respectively. Besides, the system exhibited good release behavior. DOX release rate from BM-DOX-shPlk1 was 54% after incubation with phosphate-buffered saline at 43°C and 37% after incubation with 50% fetal bovine serum, which was significantly higher than that at 37°C (P<0.05). In addition, the expressions of Plk1 mRNA and protein were significantly suppressed in cells treated with BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF compared to other groups (P<0.05). Furthermore, evaluation of the effect of in vitro antitumor revealed that BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF was significantly more effective than others in tumor inhibition. In conclusion, the new heat-sensitive co-delivery system represents a promising approach for the treatment of cancer.

Carbon nanospheres mediated delivery of nuclear matrix protein SMAR1 to direct experimental autoimmune encephalomyelitis in mice.

Owing to the suppression of immune responses and associated side effects, steroid based treatments for inflammatory encephalitis disease can be detrimental. Here, we demonstrate a novel carbon nanosphere (CNP) based treatment regime for encephalomyelitis in mice by exploiting the functional property of the nuclear matrix binding protein SMAR1. A truncated part of SMAR1 ie, the DNA binding domain was conjugated with hydrothermally synthesized CNPs. When administered intravenously, the conjugate suppressed experimental animal encephalomyelitis in T cell specific conditional SMAR1 knockout mice (SMAR(-/-)). Further, CNP-SMAR1 conjugate delayed the onset of the disease and reduced the demyelination significantly. There was a significant decrease in the production of IL-17 after re-stimulation with MOG. Altogether, our findings suggest a potential carbon nanomaterial based therapeutic intervention to combat Th17 mediated autoimmune diseases including experimental autoimmune encephalomyelitis.

Identification and Testing of Novel CARP-1 Functional Mimetic Compounds as Inhibitors of Non-Small Cell Lung and Triple Negative Breast Cancers.

The triple negative breast cancer (TNBCs) and non-small cell lung cancers (NSCLCs) often acquire mutations that contribute to failure of drugs in clinic and poor prognosis, thus presenting an urgent need to develop new and improved therapeutic modalities. Here we report that CARP-1 functional mimetic (CFMs) compounds 4 and 5, and 4.6, a structurally related analog of CFM-4, are potent inhibitors of TNBC and NSCLC cells in vitro. Cell growth suppression by CFM-4 and -4.6 involved interaction and elevated expression of CARP-1/CCAR1 and Death Effector Domain (DED) containing DNA binding (DEDD)2 proteins. Apoptosis by these compounds also involved activation of pro-apoptotic stress-activated kinases p38 and JNK1/2, cleavage of PARP and loss of mitotic cyclin B1. Both the CFMs inhibited abilities of NSCLC and TNBC cells to migrate, invade, and form colonies in suspension, while disrupting tubule formation by the human umbilical vein endothelial cells (HUVECs). Nano-lipid formulation of CFM-4 (CFM-4 NLF) enhanced its serum bioavailability when compared with the free CFM-4. Oral administration of CFM-4 NLF reduced weights and volume of the xenografted tumors derived from A549 NSCLC and MDA-MB-231 TNBC cells. Although no gross tissue or histological toxicities were noticed, the immuno-histochemical analysis revealed increased CARP-1 and DNA fragmentation in tumors of the CFM-4 NLF-treated animals. In conclusion, while stimulation of pro-apoptotic CARP-1 and DEDD2 expression and their binding underscore a novel mechanism of apoptosis transduction by CFM compounds, our proof-of-concept xenograft studies demonstrate therapeutic potential of CFM-4 for TNBC and NSCLC.

Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.

Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aß1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aß1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aß1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice.

Transscleral sustained vasohibin-1 delivery by a novel device suppressed experimentally-induced choroidal neovascularization.

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 µM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 µM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 µM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach.

Reduction of laser-induced choroidal neovascularization by intravitreal vasohibin-1 in monkey eyes.

PURPOSE: To determine whether intravitreal vasohibin-1 will reduce the grade of the choroidal neovascularization in monkey eyes. METHODS: Choroidal neovascularizations were induced in 12 monkey eyes by laser photocoagulation. Three monkeys were evaluated for the safety of the vasohibin-1 injections, 6 monkeys for the effects of a single injection, and 3 monkeys for repeated injections of vasohibin-1. Ophthalmoscopy, fluorescein angiography, focal electroretinograms, and optical coherence tomography were used for the evaluations. The level of vascular endothelial growth factor in the aqueous was determined by enzyme-linked immunosorbent assay. Immunohistochemistry was performed. RESULTS: An intravitreal injection of 10 µg of vasohibin-1 induced mild intraocular inflammation. Eyes with an intravitreal injection of 0.1 µg and 1.0 µg of vasohibin-1 had significant less fluorescein leakage from the choroidal neovascularizations and larger amplitude focal electroretinograms than that of vehicle-injected eyes. Similar results were obtained by repeated injections of 0.1 µg of vasohibin-1. Immunohistochemistry showed that vasohibin-1 was expressed mainly in the endothelial cells within the choroidal neovascularizations. The vascular endothelial growth factor level was not significantly altered by intravitreal vasohibin-1. CONCLUSION: The reduction of the laser-induced choroidal neovascularizations and preservation of macular function in monkey by intravitreal vasohibin-1 suggest that it should be considered for suppressing choroidal neovascularizations in humans.

Seeking alternative biological therapies: the future of targeted molecular treatment.

In recent years targeted therapies have expanded treatment options for patients with squamous cell cancer of the head and neck (SCCHN) considerably and have led to clinically significant benefit. However, the multitude of new targeted drugs that have emerged and are in development also represent a challenge and many years of carefully conducted clinical studies will be needed to explore their full potential. This article summarizes the most important recent developments in the targeted treatment of SCCHN and provides an overview of pathways and promising potential targets that could impact the treatment of patients with SCCHN in the future.

Hec1 overexpression hyperactivates the mitotic checkpoint and induces tumor formation in vivo.

Hec1 (Highly Expressed in Cancer 1) is one of four proteins of the outer kinetochore Ndc80 complex involved in the dynamic interface between centromeres and spindle microtubules. Its overexpression is seen in a variety of human tumors and correlates with tumor grade and prognosis. We show here that the overexpression of Hec1 in an inducible mouse model results in mitotic checkpoint hyperactivation. As previously observed with overexpression of the Mad2 gene, hyperactivation of the mitotic checkpoint leads to aneuploidy in vitro and is sufficient to generate tumors in vivo that harbor significant levels of aneuploidy. These results underscore the role of chromosomal instability as a result of mitotic checkpoint hyperactivation in the initiation of tumorigenesis.

Myocardial regeneration therapy for ischemic cardiomyopathy with cyclin A2.

OBJECTIVE: Heart failure therapies ranging from revascularization to remodeling to replacement are variably effective. Theoretically, endogenous repair via myocardial regeneration would be an ideal therapy. This study examined the ability to initiate regeneration by adenoviral-mediated expression of the cell cycle regulator cyclin A2. Our prior studies have demonstrated robust cyclin A2 transgene expression and marked antiphosphorylated histone H3 activity with this strategy, indicating the induction of cardiomyocyte mitosis. METHODS: Adult male, Lewis rats underwent left anterior descending coronary artery ligation followed by intramyocardial delivery of either cyclin A2 adenoviral vector (n = 8) or empty adeno-null vector as a control (n = 8) into the peri-infarct border zone. In vivo myocardial function was analyzed by echocardiography and invasive left ventricular pressure catheter at 6 weeks, when the animals are traditionally in heart failure. Hearts were explanted for immunoblotting and left ventricular geometric analysis. Cellular proliferation was assessed by proliferating cellular nuclear antigen expression. RESULTS: Cyclin A2 hearts exhibited improved left ventricular function as compared with controls including enhanced cardiac output (32 +/- 3.3 vs 26 +/- 5.0 mL/min, P < .05), stroke volume (0.16 +/- 0.04 vs 0.11 +/- 0.04 mL, P < .05), ejection fraction (72% +/- 7.4% vs 46.% +/- 8.5%, P < .05), fractional shortening (35% +/- 5.4% vs 19% +/- 4.3%, P < .002), maximum pressure (72 +/- 9.3 vs 61 +/- 2.9 mm Hg, P < .05), and end-systolic pressure (67 +/- 7.0 vs 55 +/- 7.0 mm Hg, P < .05). Enhanced myocardial preservation was demonstrated by enhanced left ventricular border zone wall thickness. Increased myocardial proliferation was evidenced by increased expression of proliferating cell nuclear antigen expression in cyclin A2-treated hearts. CONCLUSIONS: In failing hearts, targeted delivery of cyclin A2 improves hemodynamic function, as measured by echocardiography and pressure catheter analysis, preserves ventricular wall thickness, and may serve as an ideal myocardial regenerative therapy.