Bilirubin Nanoparticles Protect Against Cardiac Ischemia/Reperfusion Injury in Mice.

Background Ischemia/reperfusion (I/R) injury causes overproduction of reactive oxygen species, which are the major culprits of oxidative stress that leads to inflammation, apoptosis, myocardial damage, and dysfunction. Bilirubin acts as a potent endogenous antioxidant that is capable of scavenging various reactive oxygen species. We have previously generated bilirubin nanoparticles (BRNPs) consisting of polyethylene glycol-conjugated bilirubin. In this study, we examined the therapeutic effects of BRNPs on myocardial I/R injury in mice. Methods and Results In vivo imaging using fluorophore encapsulated BRNPs showed BRNPs preferentially targeted to the site of I/R injury in the heart. Cardiac I/R surgery was performed by first ligating the left anterior descending coronary artery. After 45 minutes, reperfusion was achieved by releasing the ligation. BRNPs were administered intraperitoneally at 5 minutes before and 24 hours after reperfusion. Mice that received BRNPs showed significant improvements in their cardiac output, assessed by echocardiogram and pressure volume loop measurements, compared with the ones that received vehicle treatment. BRNPs treatment also significantly reduced the myocardial infarct size in mice that underwent cardiac I/R, compared with the vehicle-treatment group. In addition, BRNPs effectively suppressed reactive oxygen species and proinflammatory factor levels, as well as the amount of cardiac apoptosis. Conclusions Taken together, BRNPs could exert their therapeutic effects on cardiac I/R injury through attenuation of oxidative stress, apoptosis, and inflammation, providing a novel therapeutic modality for myocardial I/R injury.

H2O2-Responsive Antioxidant Nanoparticle Attenuates Whole Body Ischemia/Reperfusion-Induced Multi-Organ Damages.

Mortality and morbidity after cardiac arrest remain high due to ischemia/reperfusion (I/R) injury causing multi-organ damages, even after successful return of spontaneous circulation. We previously generated H2O2-activatable antioxidant nanoparticles formulated with copolyoxalate containing vanillyl alcohol (PVAX) to prevent I/R injury. In this study, we examined whether PVAX could effectively reduce organ damages in a rat model of whole-body ischemia/reperfusion injury (WBIR). To induce a cardiac arrest, 70µl/100 g body weight of 1 mmol/l potassium chloride was administered via the jugular venous catheter. The animals in both the vehicle and PVAX-treated groups had similar baseline blood pressure. After 5.5 minutes of cardiac arrest, animals were resuscitated via intravenous epinephrine followed by chest compressions. PVAX or vehicle was injected after the spontaneous recovery of blood pressure was noted, followed by the same dose of second injection 10 minutes later. After 24 hours, multiple organs were harvested for pathological, biochemical, molecular analyses. No significant difference on the restoration of spontaneous circulation was observed between vehicle and PVAX groups. Analysis of organs harvested 24 hours post procedure showed that whole body I/R significantly increased reactive oxygen species (ROS) generation, inflammatory markers, and apoptosis in multiple organs (heart, brain, and kidney). PVAX treatment effectively blocked ROS generation, reduced the elevation of pro-inflammatory cytokines, and decreased apoptosis in these organs. Taken together, our results suggest that PVAX has potent protective effect against WBIR induced multi-organ injury, possibly by blocking ROS-mediated cell damage.

Aldosterone-Sensing Neurons in the NTS Exhibit State-Dependent Pacemaker Activity and Drive Sodium Appetite via Synergy with Angiotensin II Signaling.

Sodium deficiency increases angiotensin II (ATII) and aldosterone, which synergistically stimulate sodium retention and consumption. Recently, ATII-responsive neurons in the subfornical organ (SFO) and aldosterone-sensitive neurons in the nucleus of the solitary tract (NTSHSD2 neurons) were shown to drive sodium appetite. Here we investigate the basis for NTSHSD2 neuron activation, identify the circuit by which NTSHSD2 neurons drive appetite, and uncover an interaction between the NTSHSD2 circuit and ATII signaling. NTSHSD2 neurons respond to sodium deficiency with spontaneous pacemaker-like activity-the consequence of "cardiac" HCN and Nav1.5 channels. Remarkably, NTSHSD2 neurons are necessary for sodium appetite, and with concurrent ATII signaling their activity is sufficient to produce rapid consumption. Importantly, NTSHSD2 neurons stimulate appetite via projections to the vlBNST, which is also the effector site for ATII-responsive SFO neurons. The interaction between angiotensin signaling and NTSHSD2 neurons provides a neuronal context for the long-standing "synergy hypothesis" of sodium appetite regulation.

Neuropeptide Y3-36 incorporated into PVAX nanoparticle improves functional blood flow in a murine model of hind limb ischemia.

We generated a novel nanoparticle called PVAX, which has intrinsic antiapoptotic and anti-inflammatory properties. This nanoparticle was loaded with neuropeptide Y3-36 (NPY3-36), an angiogenic neurohormone that plays a central role in angiogenesis. Subsequently, we investigated whether PVAX-NPY3-36 could act as a therapeutic agent and induce angiogenesis and vascular remodeling in a murine model of hind limb ischemia. Adult C57BL/J6 mice (n = 40) were assigned to treatment groups: control, ischemia PBS, ischemia PVAX, ischemia NPY3-36, and Ischemia PVAX-NPY3-36 Ischemia was induced by ligation of the femoral artery in all groups except control and given relevant treatments (PBS, PVAX, NPY3-36, and PVAX-NPY3-36). Blood flow was quantified using laser Doppler imaging. On days 3 and 14 posttreatment, mice were euthanized to harvest gastrocnemius muscle for immunohistochemistry and immunoblotting. Blood flow was significantly improved in the PVAX-NPY3-36 group after 14 days. Western blot showed an increase in angiogenic factors VEGF-R2 and PDGF-ß (P = 0.0035 and P = 0.031, respectively) and antiapoptotic marker Bcl-2 in the PVAX-NPY3-36 group compared with ischemia PBS group (P = 0.023). Proapoptotic marker Smad5 was significantly decreased in the PVAX-NPY3-36 group as compared with the ischemia PBS group (P = 0.028). Furthermore, Y2 receptors were visualized in endothelial cells of newly formed arteries in the PVAX-NPY3-36 group. In conclusion, we were able to show that PVAX-NPY3-36 can induce angiogenesis and arteriogenesis as well as improve functional blood flow in a murine model of hind limb ischemia.NEW & NOTEWORTHY Our research project proposes a novel method for drug delivery. Our patented PVAX nanoparticle can detect areas of ischemia and oxidative stress. Although there have been studies about delivering angiogenic molecules to areas of ischemic injury, there are drawbacks of nonspecific delivery as well as short half-lives. Our study is unique because it can specifically deliver NPY3-36 to ischemic tissue and appears to extend the amount of time therapy is available, despite NPY3-36's short half-life.

Hydrogen Peroxide-Responsive Nanoparticle Reduces Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R), a large amount of reactive oxygen species (ROS) is produced. In particular, overproduction of hydrogen peroxide (H2O2) is considered to be a main cause of I/R-mediated tissue damage. We generated novel H2O2-responsive antioxidant polymer nanoparticles (PVAX and HPOX) that are able to target the site of ROS overproduction and attenuate the oxidative stress-associated diseases. In this study, nanoparticles were examined for their therapeutic effect on myocardial I/R injury. METHODS AND RESULTS: The therapeutic effect of nanoparticles during cardiac I/R was evaluated in mice. A single dose of PVAX (3 mg/kg) showed a significant improvement in both cardiac output and fraction shortening compared with poly(lactic-coglycolic acid) (PLGA) particle, a non-H2O2-activatable nanoparticle. PVAX also significantly reduced the myocardial infarction/area compared with PLGA (48.7±4.2 vs 14.5±2.1). In addition, PVAX effectively reduced caspase-3 activation and TUNEL-positive cells compared with PLGA. Furthermore, PVAX significantly decreased TNF-α and MCP-1 mRNA levels. To explore the antioxidant effect of PVAX by scavenging ROS, dihydroethidium staining was used as an indicator of ROS generation. PVAX effectively suppressed the generation of ROS caused by I/R, whereas a number of dihydroethidium-positive cells were observed in a group with PLGA I/R. In addition, PVAX significantly reduced the level of NADPH oxidase (NOX) 2 and 4 expression, which favors the reduction in ROS generation after I/R. CONCLUSIONS: Taken together, these results suggest that H2O2-responsive antioxidant PVAX has tremendous potential as a therapeutic agent for myocardial I/R injury.

Carboxy-terminal deletion of the HDL receptor reduces receptor levels in liver and steroidogenic tissues, induces hypercholesterolemia, and causes fatal heart disease.

The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.

Hydrogen peroxide-activatable antioxidant prodrug as a targeted therapeutic agent for ischemia-reperfusion injury.

Overproduction of hydrogen peroxide (H2O2) causes oxidative stress and is the main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury. Suppression of oxidative stress is therefore critical in the treatment of I/R injury. Here, we report H2O2-activatable antioxidant prodrug (BRAP) that is capable of specifically targeting the site of oxidative stress and exerting anti-inflammatory and anti-apoptotic activities. BRAP with a self-immolative boronic ester protecting group was designed to scavenge H2O2 and release HBA (p-hydroxybenzyl alcohol) with antioxidant and anti-inflammatory activities. BRAP exerted potent antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)- and H2O2-stimulated cells by suppressing the generation of ROS and pro-inflammatory cytokines. In mouse models of hepatic I/R and cardiac I/R, BRAP exerted potent antioxidant, anti-inflammatory and anti-apoptotic activities due to the synergistic effects of H2O2-scavenging boronic esters and therapeutic HBA. In addition, administration of high doses of BRAP daily for 7 days showed no renal or hepatic function abnormalities. Therefore BRAP has tremendous therapeutic potential as H2O2-activatable antioxidant prodrug for the treatment of I/R injuries.

Exaggerated hypertensive response to combretastatin A-4 phosphate in hypertensive rats: Effective pharmacological inhibition by diltiazem.

Combretastatin A-4 phosphate (CA4P), a tubulin depolymerizing agent, shows promise in anti-cancer therapy and is associated with dose-dependent transient hypertension. The cardiac consequence of this hypertensive effect is unknown. This study was conducted to examine the cardiotoxic effect of CA4P on a rat model of hypertension. Hypertensive rats were created by feeding a 6% high salt (HS) diet to Dahl salt sensitive (DSS) rats for 2.5weeks. Cardiac toxicity was measured using serum troponin I levels 24h after CA4P administration. In rats fed HS diet, there was a significant increase in mean arterial blood pressure (MAP) from baseline, which was further increased by 80% following CA4P administration with peak systolic blood pressure (BP) of 247mmHg. Treatment with the calcium channel blockers, diltiazem and nicardipine, completely inhibited the hypertensive effects of CA4P. Nitroglycerin or enalapril, however, failed to completely block the hypertensive effects of CA4P. CA4P injection also significantly increased the cardiac troponin I level in hypertensive rats though pretreatment with diltiazem effectively blocked troponin I increase after CA4P administration. Based on these findings, an exaggerated hypertensive response to CA4P is associated with myocardial damage in hypertensive rats. Calcium channel blockers effectively blocked both CA4P induced hypertension and cardiac damage.

p-Hydroxybenzyl alcohol-containing biodegradable nanoparticle improves functional blood flow through angiogenesis in a mouse model of hindlimb ischemia.

Therapeutic angiogenesis has achieved promising results for ischemic diseases or peripheral artery disease in preclinical and early-phase clinical studies. We examined the therapeutic angiogenic effects of HPOX, which is biodegradable polymer composing the antioxidant p-hydroxybenzyl alcohol (HBA), in a mouse model of hindlimb ischemia. HPOX effectively stimulated blood flow recovery, compared with its degraded compounds HBA and 1,4-cyclohexendimethanol, via promotion of capillary vessel density in the ischemic hindlimb. These effects were highly correlated with levels of angiogenic inducers, vascular endothelial cell growth factor (VEGF), heme oxygenase-1 (HO-1), and Akt/AMPK/endothelial nitric oxide synthase (eNOS) in ischemic mouse hindlimb muscle. Blood perfusion and neovascularization induced by HPOX were reduced in eNOS(-/-) and HO-1(+/-) mice. HPOX also elevated the endothelial cell markers VEGF receptor-2, CD31, and eNOS mRNAs in the ischemic hindlimb, indicating that HPOX increases endothelial cell population and angiogenesis in the ischemic muscle. However, this nanoparticle suppressed expression levels of several inflammatory genes in ischemic tissues. These results suggest that HPOX significantly promotes angiogenesis and blood flow perfusion in the ischemic mouse hindlimb via increased angiogenic inducers, along with suppression of inflammatory gene expression. Thus, HPOX can be used potentially as a noninvasive drug intervention to facilitate therapeutic angiogenesis.

Endothelin A receptor antagonist, atrasentan, attenuates renal and cardiac dysfunction in Dahl salt-hypertensive rats in a blood pressure independent manner.

Proteinuria is a hallmark of chronic kidney disease (CKD) and cardiovascular disease (CVD), and a good predictor of clinical outcome. Selective endothelin A (ETA) receptor antagonist used with renin-angiotensin system (RAS) inhibitors prevents development of proteinuria in CKD. However, whether the improvement in proteinuria would have beneficial effects on CVD, independent of RAS inhibition, is not well understood. In this study, we investigated whether atrasentan, an ETA receptor antagonist, has renal and cardiovascular effects independent of RAS inhibition. Male Dahl salt sensitive (DSS) rats, at six weeks of age, received water with or without different doses of atrasentan and/or enalapril under high salt (HS) diet or normal diet (ND) for 6 weeks. At the end of 12th week, atrasentan at a moderate dose significantly attenuated proteinuria and serum creatinine without reducing mean arterial pressure (MAP), thereby preventing cardiac hypertrophy and improving cardiac function. ACE inhibitor enalapril at a dose that did not significantly lowered BP, attenuated cardiac hypertrophy while moderately improving cardiac function without reducing proteinuria and serum creatinine level. Nonetheless, combined therapy of atrasentan and enalapril that does not altering BP exerted additional cardioprotective effect. Based on these findings, we conclude that BP independent monotherapy of ETA receptor antagonist attenuates the progression of CKD and significantly mitigates CVD independent of RAS inhibition.

A new Mdr2(-/-) mouse model of sclerosing cholangitis with rapid fibrosis progression, early-onset portal hypertension, and liver cancer.

We previously characterized the Mdr2(Abcb4)(-/-) mouse as a reproducible model of chronic biliary liver disease. However, it demonstrates relatively slow fibrosis progression, possibly due to its fibrosis-resistant genetic background. We aimed to improve the model by moving it onto a fibrosis-susceptible background. We generated novel BALB/c.Mdr2(-/-) mouse via genetic backcross onto highly fibrosis-susceptible BALB/c substrain, identified in inbred mouse strain screening. Liver fibrosis, portal pressure, and hepatic tumor burden in BALB/c.Mdr2(-/-) mice were studied up to 1 year of age in direct comparison to parental strain FVB.Mdr2(-/-). BALB/c.Mdr2(-/-) mice developed periductular onion-skin type fibrotic lesions and pronounced ductular reaction starting from 4 weeks of age. Compared to parental strain, BALB/c.Mdr2(-/-) mice demonstrated dramatically accelerated liver fibrosis, with threefold increase in collagen deposition and bridging fibrosis/early signs of cirrhosis at 12 weeks. This was accompanied by early-onset severe portal hypertension and twofold to fourfold increase in profibrogenic transcripts Col1a1 [procollagen α1(I)], Tgfb1, and Timp1. Primary liver cancers in BALB/c.Mdr2(-/-) developed earlier, with greater tumor burden compared to FVB.Mdr2(-/-). BALB/c.Mdr2(-/-) mice have unprecedented degree and rapidity of hepatic fibrosis progression and clinically relevant cirrhosis complications, such as early-onset portal hypertension and primary liver cancers. This new model will facilitate development of antifibrotic drugs and studies into mechanisms of biliary fibrosis progression.

RhoA signaling in cardiomyocytes protects against stress-induced heart failure but facilitates cardiac fibrosis.

The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis).

Abnormal calcium handling and exaggerated cardiac dysfunction in mice with defective vitamin d signaling.

AIM: Altered vitamin D signaling is associated with cardiac dysfunction, but the pathogenic mechanism is not clearly understood. We examine the mechanism and the role of vitamin D signaling in the development of cardiac dysfunction. METHODS AND RESULTS: We analyzed 1α-hydroxylase (1α-OHase) knockout (1α-OHase-/-) mice, which lack 1α-OH enzymes that convert the inactive form to hormonally active form of vitamin D. 1α-OHase-/- mice showed modest cardiac hypertrophy at baseline. Induction of pressure overload by transverse aortic constriction (TAC) demonstrated exaggerated cardiac dysfunction in 1α-OHase-/- mice compared to their WT littermates with a significant increase in fibrosis and expression of inflammatory cytokines. Analysis of calcium (Ca2+) transient demonstrated profound Ca2+ handling abnormalities in 1α-OHase-/- mouse cardiomyocytes (CMs), and treatment with paricalcitol (PC), an activated vitamin D3 analog, significantly attenuated defective Ca2+ handling in 1α-OHase-/- CMs. We further delineated the effect of vitamin D deficiency condition to TAC by first correcting the vitamin D deficiency in 1α-OHase-/- mice, followed then by either a daily maintenance dose of vitamin D or vehicle (to achieve vitamin D deficiency) at the time of sham or TAC. In mice treated with vitamin D, there was a significant attenuation of TAC-induced cardiac hypertrophy, interstitial fibrosis, inflammatory markers, Ca2+ handling abnormalities and cardiac function compared to the vehicle treated animals. CONCLUSIONS: Our results provide insight into the mechanism of cardiac dysfunction, which is associated with severely defective Ca2+ handling and defective vitamin D signaling in 1α-OHase-/- mice.

Therapeutic use of H2O2-responsive anti-oxidant polymer nanoparticles for doxorubicin-induced cardiomyopathy.

Doxorubicin (DOX) is a commonly used anti-neoplastic agent but its clinical use is limited due to serious hepatic and cardiac side effects. DOX-induced toxicity is mainly associated with overproduction of reactive species oxygen (ROS) such as hydrogen peroxide (H2O2). We have recently developed H2O2-responsive anti-oxidant polymer, polyoxalate containing vanillyl alcohol (PVAX), which is designed to rapidly scavenge H2O2 and release vanillyl alcohol with anti-oxidant, anti-inflammatory and anti-apoptotic properties. In this study, we report that PVAX nanoparticles are novel therapeutic agents for treating DOX-induced cardiac and hepatic toxicity. Intraperitoneal injection of PVAX nanoparticles (4 mg/kg/day) resulted in significant inhibition in apoptosis in liver and heart of DOX-treated mice by suppressing the activation of poly (ADP ribose) polymerase 1 (PARP-1) and caspase-3. PVAX treatment also prevented DOX-induced cardiac dysfunction. Furthermore, survival rate (vehicle = 35% vs. PVAX = 75%; p < 0.05) was significantly improved in a PVAX nanoparticles-treated group compared with vehicle treated groups. Taken together, we anticipate that PVAX nanoparticles could be a highly specific and potent treatment modality in DOX-induced cardiac and hepatic toxicity.

Hydrogen peroxide-responsive copolyoxalate nanoparticles for detection and therapy of ischemia-reperfusion injury.

The main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury is the generation of high level of hydrogen peroxide (H2O2). In this study, we report a novel diagnostic and therapeutic strategy for I/R injury based on H2O2-activatable copolyoxalate nanoparticles using a murine model of hind limb I/R injury. The nanoparticles are composed of hydroxybenzyl alcohol (HBA)-incorporating copolyoxalate (HPOX) that, in the presence of H2O2, degrades completely into three known and safe compounds, cyclohexanedimethanol, HBA and CO2. HPOX effectively scavenges H2O2 in a dose-dependent manner and hydrolyzes to release HBA which exerts intrinsic antioxidant and anti-inflammatory activities both in vitro and in vivo models of hind limb I/R. HPOX nanoparticles loaded with fluorophore effectively and robustly image H2O2 generated in hind limb I/R injury, demonstrating their potential for bioimaging of H2O2-associated diseases. Furthermore, HPOX nanoparticles loaded with anti-apoptotic drug effectively release the drug payload after I/R injury, exhibiting their effectiveness for a targeted drug delivery system for I/R injury. We anticipate that multifunctional HPOX nanoparticles have great potential as H2O2 imaging agents, therapeutics and drug delivery systems for H2O2-associated diseases.

H2O2-responsive molecularly engineered polymer nanoparticles as ischemia/reperfusion-targeted nanotherapeutic agents.

The main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury is the overproduction of reactive oxygen species (ROS). Hydrogen peroxide (H2O2), the most abundant form of ROS produced during I/R, causes inflammation, apoptosis and subsequent tissue damages. Here, we report H2O2-responsive antioxidant nanoparticles formulated from copolyoxalate containing vanillyl alcohol (VA) (PVAX) as a novel I/R-targeted nanotherapeutic agent. PVAX was designed to incorporate VA and H2O2-responsive peroxalate ester linkages covalently in its backbone. PVAX nanoparticles therefore degrade and release VA, which is able to reduce the generation of ROS, and exert anti-inflammatory and anti-apoptotic activity. In hind-limb I/R and liver I/R models in mice, PVAX nanoparticles specifically reacted with overproduced H2O2 and exerted highly potent anti-inflammatory and anti-apoptotic activities that reduced cellular damages. Therefore, PVAX nanoparticles have tremendous potential as nanotherapeutic agents for I/R injury and H2O2-associated diseases.

Doxercalciferol, a pro-hormone of vitamin D, prevents the development of cardiac hypertrophy in rats.

BACKGROUND: Activated vitamin D analog, paricalcitol, has been shown to attenuate the development of cardiac hypertrophy in Dahl salt sensitive (DSS) rats. To determine whether an antihypertrophic effect is class specific, we tested if doxercalciferol (a pro-hormone vitamin D2 analog) could also attenuate the development of cardiac hypertrophy in DSS rats. METHODS AND RESULTS: Male DSS rats were fed a high salt (HS) diet for 6 weeks beginning at 6 weeks of age. Doxercalciferol was administered intraperitoneally at 150 ng, 3 times per week (Monday, Wednesday, Friday) for 6 weeks. Pathological and echocardiographic findings demonstrated that rats on HS diet with doxercalciferol administration had significant decrease in cardiac hypertrophy and improved cardiac function compared to the HS + vehicle. In addition, there was a significant decrease in plasma brain natriuretic peptide (BNP) level and tissue atrial natriuretic factor (ANF) mRNA level with doxercalciferol treatment. Doxercalciferol also significantly reduced the level of protein kinase C-α (PKCα) suggesting that PKC-mediated cardiac hypertrophy may be associated with vitamin D deficiency. CONCLUSIONS: Administration of doxercalciferol attenuated the development of HS diet induced cardiac hypertrophy and cardiac dysfunction in DSS rats.

Preventing progression of cardiac hypertrophy and development of heart failure by paricalcitol therapy in rats.

AIMS: Vitamin D deficiency is associated with cardiac hypertrophy and heart failure, and vitamin D therapy prevents the progression of cardiac hypertrophy in animal models. Here, we examine whether vitamin D therapy prevents progression of pre-existing cardiac hypertrophy and development of heart failure. METHODS AND RESULTS: When male Dahl salt-sensitive rats were fed a high salt (HS) diet, all rats developed cardiac hypertrophy after 5 weeks. Thereafter, rats were treated with vehicle (V), paricalcitol (PC, an active vitamin D analogue, at 200 ng, IP 3x/week), enalapril (EP, 90 µg/day), and PC + EP. All groups were continued on the HS diet and evaluated after 4 weeks of therapy. The PC and PC + EP groups, but not the V and EP only groups, showed significant prevention of progression of pre-existing cardiac hypertrophy. The signs of decompensated heart failure were evident in the vehicle-treated group; these heart failure parameters significantly improved with PC, EP or PC + EP therapy. The expression of PKCα, which is regulated by Ca(2+)and known to stimulate cardiac hypertrophy, was significantly increased in the vehicle group, and PC, EP or PC + EP effectively decreased PKCα activation. We also observed normalization of genetic alterations during progression to heart failure with PC treatment. CONCLUSION: PC treatment resulted in both the prevention of progression of pre-existing cardiac hypertrophy and the development of heart failure, compared with improvement in progression to heart failure by EP alone. These beneficial findings in heart were associated with inhibition of PKCα activation and reversal of gene alterations.

Mitochondria to nucleus translocation of AIF in mice lacking Hsp70 during ischemia/reperfusion.

Heat shock protein 70 (Hsp70) has been shown to have an anti-apoptotic function, but its mechanism is not clear in heart. In this study, we examined the effect of Hsp70 deletion on AIF-induced apoptosis during ischemia/reperfusion (I/R) in vivo. Although Hsp70 KO and WT mice demonstrated similar amounts of AIF released from mitochondria after I/R surgery, Hsp70 KO mice showed a significantly greater increase in apoptosis, larger infarct size, and decreased cardiac output. There was also a significant fourfold increase in the nuclear accumulation of AIF in Hsp70 KO mice compared with WT mice. Treatment with 4-AN (4-amino-1,8-napthalimide, 3 mg/kg), a potent inhibitor of PARP-1, which is a critical regulator of AIF-induced apoptosis, significantly blocked the release of AIF from mitochondria and the translocation of AIF into the nuclei after I/R in both WT and Hsp70 KO mice. In addition, 4-AN treatment resulted in a significant inhibition of apoptosis, a reduction of infarct size, and attenuated cardiac dysfunction in both WT and Hsp70 KO mice after I/R. The anti-apoptotic function of Hsp70 occurs through the inhibition of AIF-induced apoptosis by blocking the mitochondria to nucleus translocation of AIF. PARP-1 inhibition improves cardiac function by blocking AIF-induced apoptosis.

Delayed activation of caspase-independent apoptosis during heart failure in transgenic mice overexpressing caspase inhibitor CrmA.

Although caspase activation is generally thought to be necessary to induce apoptosis, recent evidence suggests that apoptosis can be activated in the setting of caspase inhibition. In this study, we tested the hypothesis that caspase-independent apoptotic pathways contribute to the development of heart failure in the absence of caspase activation. Acute cardiomyopathy was induced using a single dose of doxorubicin (Dox, 20 mg/kg) injected into male wild-type (WT) and transgenic (Tg) mice with a cardiac-specific expression of cytokine response modifier A (CrmA), a known caspase inhibitor. Early (6 day) survival was significantly better in CrmA Tg (81%) than WT (38%) mice. Twelve days after Dox injection, however, the mortality benefit had dissipated, and increased cardiac apoptosis was observed in both groups. There was, however, a significantly greater release of apoptosis-inducing factor (AIF) from mitochondria to cytosol in CrmA Tg compared with WT mice, which suggests that an enhancement of activation in caspase-independent apoptotic pathways had occurred. The administration of a poly(ADP-ribose) polymerase-1 inhibitor, 4-amino-1,8-naphthalimide (4-AN), to Dox-treated mice resulted in significantly improved cardiac function, a significant blockade of AIF released from mitochondria, and decreased cardiac apoptosis. There were also significantly improved survival in WT (18% without 4-AN vs. 89% with 4-AN) and CrmA Tg (13% without 4-AN vs. 93% with 4-AN) mice 12 days after Dox injection. In conclusion, these findings suggest that apoptosis can be induced in the heart lacking caspase activation via caspase-independent pathways and that enabling the inhibition of AIF activation may provide a significant cardiac benefit.