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.

The Prominin-1-Derived Peptide Improves Cardiac Function Following Ischemia.

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.

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.

Oxidative Stress and Antioxidant Treatments in Cardiovascular Diseases.

Oxidative stress plays a key role in many physiological and pathological conditions. The intracellular oxidative homeostasis is tightly regulated by the reactive oxygen species production and the intracellular defense mechanisms. Increased oxidative stress could alter lipid, DNA, and protein, resulting in cellular inflammation and programmed cell death. Evidences show that oxidative stress plays an important role in the progression of various cardiovascular diseases, such as atherosclerosis, heart failure, cardiac arrhythmia, and ischemia-reperfusion injury. There are a number of therapeutic options to treat oxidative stress-associated cardiovascular diseases. Well known antioxidants, such as nutritional supplements, as well as more novel antioxidants have been studied. In addition, novel therapeutic strategies using miRNA and nanomedicine are also being developed to treat various cardiovascular diseases. In this article, we provide a detailed description of oxidative stress. Then, we will introduce the relationship between oxidative stress and several cardiovascular diseases. Finally, we will focus on the clinical implications of oxidative stress in cardiovascular diseases.

Neuropeptide Y3-36 incorporated into PVAX nanoparticle improves angiogenesis in a murine model of myocardial ischemia.

Neuropeptide-Y (NPY) leads to angiogenesis and remodeling of the ischemic myocardium. The objective of this study is to assess the therapeutic potential of NPY in a model of acute myocardial ischemia using a nanoparticles delivery system targeted to tissue with oxidative stress. NPY3-36 was loaded onto copolyoxalate containing vanillyl alcohol (PVAX) using a double emulsification strategy. Adult C57BL/J6 mice (n = 49) were randomly divided into PVAX-NPY3-36 (n = 22), Vehicle (Saline) (n = 16), and Sham (n = 11) groups. The ischemia to left anterior descending artery was induced in PVAX-NPY3-36 or vehicle groups. The tissue was collected at the end of two weeks after assessing the functional and echocardiographic data. There was a significant decrease in infarction size and mortality in PVAX-NPY3-36 group compared to the Vehicle group (P = 0.01 and P = 0.05). On echocardiography, there was significant improvement in contractility and diastolic parameters (P = 0.01). On pressure-volume loop there was significant increase in stroke volume (P = 0.01), cardiac output (P = 0.01) and ventricular stroke work (P = 0.01) in the PVAX-NPY3-36 group. On Western blot analysis, there was a significant increase in pro-angiogenic factors Ang-1, TGF-ß, PDGF- ß and its receptors and VEGF in the ischemic tissue treated with PVAX-NPY3-36 as compared to Vehicle ischemic tissue (P = 0.01, P = 0.0003, and P < 0.05 respectively). It may be possible to have targeted delivery of labile neurotransmitters NPY3-36 to the ischemic myocardium using nanoparticle PVAX and achieving angiogenesis and significant functional improvement.

Systemic Review of Biodegradable Nanomaterials in Nanomedicine.

BACKGROUND: Nanomedicine is a field of science that uses nanoscale materials for the diagnosis and treatment of human disease. It has emerged as an important aspect of the therapeutics, but at the same time, also raises concerns regarding the safety of the nanomaterials involved. Recent applications of functionalized biodegradable nanomaterials have significantly improved the safety profile of nanomedicine. OBJECTIVE: Our goal is to evaluate different types of biodegradable nanomaterials that have been functionalized for their biomedical applications. METHOD: In this review, we used PubMed as our literature source and selected recently published studies on biodegradable nanomaterials and their applications in nanomedicine. RESULTS: We found that biodegradable polymers are commonly functionalized for various purposes. Their property of being naturally degraded under biological conditions allows these biodegradable nanomaterials to be used for many biomedical purposes, including bio-imaging, targeted drug delivery, implantation and tissue engineering. The degradability of these nanoparticles can be utilized to control cargo release, by allowing efficient degradation of the nanomaterials at the target site while maintaining nanoparticle integrity at off-target sites. CONCLUSION: While each biodegradable nanomaterial has its advantages and disadvantages, with careful design and functionalization, biodegradable nanoparticles hold great future in nanomedicine.

Targeting of intragraft reactive oxygen species by APP-103, a novel polymer product, mitigates ischemia/reperfusion injury and promotes the survival of renal transplants.

Inflammatory responses associated with ischemia/reperfusion injury (IRI) play a central role in alloimmunity and transplant outcomes. A key event driving these inflammatory responses is the burst of reactive oxygen species (ROS), with hydrogen peroxide (H2 O2 ) as the most abundant form that occurs as a result of surgical implantation of the donor organ. Here, we used a syngeneic rat renal transplant and IRI model to evaluate the therapeutic properties of APP-103, a polyoxalate-based copolymer molecule containing vanillyl alcohol (VA) that exhibits high sensitivity and specificity toward the production of H2 O2 . We show that APP-103 is safe, and that it effectively promotes kidney function following IRI and survival of renal transplants. APP-103 reduces tissue injury and IRI-associated inflammatory responses in models of both warm ischemia (kidney clamping) and prolonged cold ischemia (syngeneic renal transplant). Mechanistically, we demonstrate that APP-103 exerts protective effects by specifically targeting the production of ROS. Our data introduce APP-103 as a novel, nontoxic, and site-activating therapeutic approach that effectively ameliorates the consequences of IRI in solid organ transplantation.

Guidelines for evaluating myocardial cell death.

Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms.

Targeting Oxidative Stress Using Nanoparticles as a Theranostic Strategy for Cardiovascular Diseases.

SIGNIFICANCE: Nanomedicine is an application of nanotechnology that provides solutions to unmet medical challenges. The unique features of nanoparticles, such as their small size, modifiable components, and diverse functionality, make them attractive and suitable materials for novel diagnostic, therapeutic, or theranostic applications. Cardiovascular diseases (CVDs) are the major cause of noncommunicable illness in both developing and developed countries. Nanomedicine offers novel theranostic options for the treatment of CVDs. Recent Advances: Many innovative nanoparticles to target reactive oxygen species (ROS) have been developed. In this article, we review the characteristics of nanoparticles that are responsive to ROS, their limitations, and their potential clinical uses. Significant advances made in diagnosis of atherosclerosis and treatment of acute coronary syndrome using nanoparticles are discussed. CRITICAL ISSUES: Although there is a tremendous potential for the nanoparticle applications in medicine, their safety should be considered while using in humans. We discuss the challenges that may be encountered with some of the innovative nanoparticles used in CVDs. FUTURE DIRECTIONS: The unique properties of nanoparticles offer novel diagnostic tool and potential therapeutic strategies. However, nanomedicine is still in its infancy, and further in-depth studies are needed before wide clinical application is achieved.

Highly conductive, stretchable and biocompatible Ag-Au core-sheath nanowire composite for wearable and implantable bioelectronics.

Wearable and implantable devices require conductive, stretchable and biocompatible materials. However, obtaining composites that simultaneously fulfil these requirements is challenging due to a trade-off between conductivity and stretchability. Here, we report on Ag-Au nanocomposites composed of ultralong gold-coated silver nanowires in an elastomeric block-copolymer matrix. Owing to the high aspect ratio and percolation network of the Ag-Au nanowires, the nanocomposites exhibit an optimized conductivity of 41,850 S cm-1 (maximum of 72,600 S cm-1). Phase separation in the Ag-Au nanocomposite during the solvent-drying process generates a microstructure that yields an optimized stretchability of 266% (maximum of 840%). The thick gold sheath deposited on the silver nanowire surface prevents oxidation and silver ion leaching, making the composite biocompatible and highly conductive. Using the nanocomposite, we successfully fabricate wearable and implantable soft bioelectronic devices that can be conformally integrated with human skin and swine heart for continuous electrophysiological recording, and electrical and thermal stimulation.

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.

Identification of Novel Non-secosteroidal Vitamin D Receptor Agonists with Potent Cardioprotective Effects and devoid of Hypercalcemia.

Vitamin D regulates many biological processes, but its clinical utility is limited by its hypercalcemic effect. Using a virtual screening platform to search novel chemical probes that activate the vitamin D signaling, we report discovery of novel non-steroidal small-molecule compounds that activate the vitamin D receptor (VDR), but are devoid of hypercalcemia. A lead compound (known as VDR 4-1) demonstrated potent transcriptional activities in a VDR reporter gene assay, and significantly ameliorated cardiac hypertrophy in cell culture studies and in animal models. VDR 4-1 also effectively suppressed secondary hyperparathyroidism in 1α-hydroxylase knockout mice. In contrast to 1α,25-dihydroxyvitamin D3 (1,25-D3 or calcitriol), a naturally occurring VDR agonist, VDR 4-1 therapy even at high doses did not induce hypercalcemia. These findings were accompanied by a lack of upregulation of calcium transport genes in kidney and in the gut providing a mechanism for the lack of hypercalcemia. Furthermore, VDR 4-1 therapy significantly suppressed cardiac hypertrophy and progression to heart failure in both vitamin D deficient and normal mice without inducing significant hypercalcemia. In conclusion, we have identified a unique VDR agonist compound with beneficial effects in mouse models of hyperparathyroidism and heart failure without inducing significant hypercalcemia.

Early Actions of Anti-Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on Angiogenic Blood Vessels.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

Fibrin-Targeted and H2O2-Responsive Nanoparticles as a Theranostics for Thrombosed Vessels.

A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H2O2), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H2O2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl3)-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis.

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.

H2O2-responsive antioxidant polymeric nanoparticles as therapeutic agents for peripheral arterial disease.

Peripheral artery disease (PAD) is a common circulatory disorder in which narrowed arteries limit blood flow to the lower extremity and affect millions of people worldwide. Therapeutic angiogenesis has emerged as a promising strategy to treat PAD patients because surgical intervention has been showing limited success. Leg muscles of PAD patients have significantly high level of ROS (reactive oxygen species) and the increased production of ROS is a key mechanism of initiation and progression of PAD. We have recently developed H2O2-responsive polymer PVAX, which is designed to rapidly scavenge H2O2 and release vanillyl alcohol with antioxidant and anti-inflammatory activity. In this study, we investigated the therapeutic efficacy of PVAX nanoparticles for PAD using a cell culture model and a mouse model of hindlimb ischemia. PVAX nanoparticles significantly enhanced the expression of angiogenic inducers such as vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule (PECAM)-1 in human umbilical vein endothelial cells (HUVEC). PVAX nanoparticles promoted revascularization and restoration of blood perfusion into ischemic tissues by upregulating angiogenic VEGF and PECAM-1. This work demonstrates that H2O2-responsive PVAX nanoparticles facilitate therapeutic angiogenesis and hold tremendous translational potential as therapeutic systems for ischemic diseases such as PAD.

Soluble fms-like tyrosine kinase 1 promotes angiotensin II sensitivity in preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy in which patients develop profound sensitivity to vasopressors, such as angiotensin II, and is associated with substantial morbidity for the mother and fetus. Enhanced vasoconstrictor sensitivity and elevations in soluble fms-like tyrosine kinase 1 (sFLT1), a circulating antiangiogenic protein, precede clinical signs and symptoms of preeclampsia. Here, we report that overexpression of sFlt1 in pregnant mice induced angiotensin II sensitivity and hypertension by impairing endothelial nitric oxide synthase (eNOS) phosphorylation and promoting oxidative stress in the vasculature. Administration of the NOS inhibitor l-NAME to pregnant mice recapitulated the angiotensin sensitivity and oxidative stress observed with sFlt1 overexpression. Sildenafil, an FDA-approved phosphodiesterase 5 inhibitor that enhances NO signaling, reversed sFlt1-induced hypertension and angiotensin II sensitivity in the preeclampsia mouse model. Sildenafil treatment also improved uterine blood flow, decreased uterine vascular resistance, and improved fetal weights in comparison with untreated sFlt1-expressing mice. Finally, sFLT1 protein expression inversely correlated with reductions in eNOS phosphorylation in placental tissue of human preeclampsia patients. These data support the concept that endothelial dysfunction due to high circulating sFLT1 may be the primary event leading to enhanced vasoconstrictor sensitivity that is characteristic of preeclampsia and suggest that targeting sFLT1-induced pathways may be an avenue for treating preeclampsia and improving fetal outcomes.

A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity.

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.