Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer.

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90 % along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.

Pitx2 prevents susceptibility to atrial arrhythmias by inhibiting left-sided pacemaker specification.

Atrial fibrillation (AF), the most prevalent sustained cardiac arrhythmia, often coexists with the related arrhythmia atrial flutter (AFL). Limitations in effectiveness and safety of current therapies make an understanding of the molecular mechanism underlying AF more urgent. Genome-wide association studies implicated a region of human chromosome 4q25 in familial AF and AFL, approximately 150 kb distal to the Pitx2 homeobox gene, a developmental left-right asymmetry (LRA) gene. To investigate the significance of the 4q25 variants, we used mouse models to investigate Pitx2 in atrial arrhythmogenesis directly. When challenged by programmed stimulation, Pitx2(null+/-) adult mice had atrial arrhythmias, including AFL and atrial tachycardia, indicating that Pitx2 haploinsufficiency predisposes to atrial arrhythmias. Microarray and in situ studies indicated that Pitx2 suppresses sinoatrial node (SAN)-specific gene expression, including Shox2, in the left atrium of embryos and young adults. In vivo ChIP and transfection experiments indicated that Pitx2 directly bound Shox2 in vivo, supporting the notion that Pitx2 directly inhibits the SAN-specific genetic program in left atrium. Our findings implicate Pitx2 and Pitx2-mediated LRA-signaling pathways in prevention of atrial arrhythmias.

Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia.

Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.

Effect of dietary palm olein oil on oxidative stress associated with ischemic-reperfusion injury in isolated rat heart.

BACKGROUND: Palm olein oil (PO), obtained from refining of palm oil is rich in monounsaturated fatty acid and antioxidant vitamins and is widely used as oil in diet in many parts of the world including India. Palm oil has been reported to have beneficial effects in oxidative stress associated with hypertension and arterial thrombosis. Oxidative stress plays a major role in the etiopathology of myocardial ischemic-reperfusion injury (IRI) which is a common sequel of ischemic heart disease. Antioxidants have potent therapeutic effects on both ischemic heart disease and ischemic-reperfusion injury. Information on the effect of PO on ischemic-reperfusion injury is, however, lacking. In the present study, the effect of dietary palm olein oil on oxidative stress associated with IRI was investigated in an isolated rat heart model. Wistar rats (150-200 gm) of either sex were divided into three different groups (n = 16). Rats were fed with palm olein oil supplemented commercial rat diet, in two different doses [5% v / w (PO 5) and 10% v / w (PO 10) of diet] for 30 days. Control rats (C) were fed with normal diet. After 30 days, half the rats from each group were subjected to in vitro myocardial IRI (20 min of global ischemia, followed by 40 min of reperfusion). Hearts from all the groups were then processed for biochemical and histopathological studies. One way ANOVA followed by Bonferroni test was applied to test for significance and values are expressed as mean +/- SE (p < 0.05). RESULTS: There was a significant increase in myocardial catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities with no significant change in myocardial thiobarbituric acid reactive substances (TBARS) only in group PO 5 as compared to group C. There was no light microscopic evidence of tissue injury. A significant rise in myocardial TBARS and depletion of myocardial endogenous antioxidants (SOD, CAT and GPx) along with significant myocyte injury was observed in control rats subjected to ischemia-reperfusion (C IR). Hearts from palm olein oil fed rats subjected to ischemia-reperfusion (PO 5 IR and PO 10 IR) were protected from increase in TBARS and depletion of endogenous antioxidants as compared to C IR group. No significant myocyte injury was present in the treated groups. CONCLUSIONS: The present study demonstrated for the first time that dietary palm olein oil protected rat heart from oxidative stress associated with ischemic-reperfusion injury.

Chronic stress impairs collateral blood flow recovery in aged mice.

Chronic stress is associated with increased risk of cardiovascular diseases. Aging is also associated with vascular dysfunction. We hypothesize that chronic stress accelerates collateral dysfunction in old mice. Mice were subjected to either chronic social defeat (CSD) or chronic cold stress (CCS). The CSD mice were housed in a box inside an aggressor's cage and exposed to the aggressor. The CCS group was placed in iced water. After chronic stress, mice underwent femoral artery ligation (FAL) and flow recovery was measured. For the CSD group, appearance and use scores of the foot and a behavioral test were performed. CSD impaired collateral flow recovery after FAL. Further, stressed mice had greater ischemic damage, impaired foot function, and altered behavior. The CCS mice also showed impaired collateral flow recovery. Chronic stress causes hind limb collateral dysfunction in old mice, a conclusion reinforced by the fact that two types of stress produced similar changes.

Donor site healing dynamics: molecular, histological, and noninvasive imaging assessment in a porcine model.

Understanding the physiology of donor site healing will lead to advances in how these wounds are treated and may ultimately allow faster healing, more frequent autografting, and more effective care of the burn-injured patient. Unfortunately, a paucity of data exists regarding perfusion metrics over the course of donor site healing. Furthermore, there are no studies that interrelate indices of perfusion with the molecular and cellular processes of donor site healing. Male Duroc pigs were anesthetized and donor site wounds were created using a Zimmer dermatome at a depth of 0.060 inch (1.52 mm). Digital photographs, laser Doppler images, and punch biopsies were obtained before and after excision and on days 2, 4, 7, 9, 11, 14, and 16 until wounds were healed. RNA isolation was performed and quantitative polymerase chain reaction was used to examine differential gene expression over the time course. Formalin-fixed biopsies were embedded in paraffin, sectioned, stained, and examined. Wound surfaces were 83% re-epithelialized by day 16. Perfusion peaked on day 2 then declined, but it remained significantly elevated compared to before excision (P < .05). From day 9 onward, mean perfusion units were not significantly different from baseline (P < .05). Twenty-two representative genes were selected for examination. RNA expression of collagen, tenascin-cytoactin, inflammatory cytokines, remodeling enzymes, growth factors, and Wnt was increased. Inflammatory cells and cytokines were demonstrated histologically. Nuclei per high powered field peaked at day 7 and neodermal thickness increased daily to day 14. A novel porcine model for donor site wound healing that interrelates re-epithelilaizationand perfusion with molecular and cellular indices has been demonstrated.

A new murine model of stress-induced complex atherosclerotic lesions.

The primary purpose of this investigation was to determine whether ApoE(-/-) mice, when subjected to chronic stress, exhibit lesions characteristic of human vulnerable plaque and, if so, to determine the time course of such changes. We found that the lesions were remarkably similar to human vulnerable plaque, and that the time course of lesion progression raised interesting insights into the process of plaque development. Lard-fed mixed-background ApoE(-/-) mice exposed to chronic stress develop lesions with large necrotic core, thin fibrous cap and a high degree of inflammation. Neovascularization and intraplaque hemorrhage are observed in over 80% of stressed animals at 20 weeks of age. Previously described models report a prevalence of only 13% for neovascularization observed at a much later time point, between 36 and 60 weeks of age. Thus, our new stress-induced model of advanced atherosclerotic plaque provides an improvement over what is currently available. This model offers a tool to further investigate progression of plaque phenotype to a more vulnerable phenotype in humans. Our findings also suggest a possible use of this stress-induced model to determine whether therapeutic interventions have effects not only on plaque burden, but also, and importantly, on plaque vulnerability.

Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling.

Despite positive animal studies, clinical angiogenesis trials have been disappointing, possibly due to risk factors present in humans but usually unexplored in animals. We recently demonstrated aging causes impaired collateral remodeling and collateral dropout; here, we investigate potential mechanisms responsible for these findings. Four-, 10-, and 18-month-C57BL/6J mice were subjected to femoral artery ligation; flow was measured using laser Doppler perfusion imaging. Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS were measured in calf muscle. Apoptosis was assessed in endothelial (EC) and smooth muscle (SMC) cells isolated from young and old mice. Angiogenesis was measured using a Matrigel plug assay. Lethally irradiated young and old mice received bone marrow cells (BMC) from either young or old donors and were subjected to femoral artery ligation (FAL). BMC mobilization and homing were assessed. Flow recovery was impaired and less eNOS and phosphorylated eNOS was present in older vs. young mice (p < 0.001 and p = 0.015, respectively). ECs and SMCs from older mice were more sensitive to an apoptotic stimulus, but were rescued by NO-enhancing drugs. In older mice, angiogenesis (Matrigel plug assay) was impaired, as was mobilization and homing of BM progenitor cells following FAL. Although both mobilization and homing improved when older mice received BMC transplantation from young donors, flow recovery failed to improve. Aging impairs BMC mobilization and homing, collateral responsiveness to angiogenic stimuli, and increases EC and SMC susceptibility to apoptosis via dysfunctional eNOS signaling. The latter could contribute to impaired remodeling and collateral dropout. These finding identify potential obstacles to therapeutic interventions in elderly patients.

Sudden infant death syndrome in mice with an inherited mutation in RyR2.

BACKGROUND: Mutations in the cardiac ryanodine receptor gene (RyR2) have been recently identified in victims of sudden infant death syndrome. The aim of this study was to determine whether a gain-of-function mutation in RyR2 increases the propensity to cardiac arrhythmias and sudden death in young mice. METHODS AND RESULTS: Incidence of sudden death was monitored prospectively in heterozygous knock-in mice with mutation R176Q in RyR2 (R176Q/+). Young R176Q/+ mice exhibited a higher incidence of sudden death compared with wild-type littermates. Optical mapping of membrane potentials and intracellular calcium in 1- to 7-day-old R176Q/+ and wild-type mice revealed an increased incidence of ventricular ectopy and spontaneous calcium releases in neonatal R176Q/+ mice. Surface ECGs in 3- to 10-day-old mice showed that R176Q/+ mice developed more ventricular arrhythmias after provocation with epinephrine and caffeine. Intracardiac pacing studies in 12- to 18-day-old mice revealed the presence of an arrhythmogenic substrate in R176Q/+ compared with wild-type mice. Reverse transcription-polymerase chain reaction and Western blotting showed that expression levels of other calcium handling proteins were unaltered, suggesting that calcium leak through mutant RyR2 underlies arrhythmogenesis and sudden death in young R176Q/+ mice. CONCLUSIONS: Our findings demonstrate that a gain-of-function mutation in RyR2 confers an increased risk of cardiac arrhythmias and sudden death in young mice and that young R176Q/+ mice may be used as a model for elucidating the complex interplay between genetic and environmental risk factors associated with sudden infant death syndrome.

Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.

BACKGROUND: Although defective Ca(2+) homeostasis may contribute to arrhythmogenesis in atrial fibrillation (AF), the underlying molecular mechanisms remain poorly understood. Studies in patients with AF revealed that impaired diastolic closure of sarcoplasmic reticulum (SR) Ca(2+)-release channels (ryanodine receptors, RyR2) is associated with reduced levels of the RyR2-inhibitory subunit FKBP12.6. OBJECTIVE: The objective of the present study was to test the hypothesis that Ca(2+) leak from the SR through RyR2 increases the propensity for AF in FKBP12.6-deficient (-/-) mice. METHODS: Surface electrocardiogram and intracardiac electrograms were recorded simultaneously in FKBP12.6-/- mice and wild-type (WT) littermates. Right atrial programmed stimulation was performed before and after injection of RyR2 antagonist tetracaine (0.5 mg/kg). Intracellular Ca(2+) transients were recorded in atrial myocytes from FKBP12.6-/- and WT mice. RESULTS: FKBP12.6-/- mice had structurally normal atria and unaltered expression of key Ca(2+)-handling proteins. AF episodes were inducible in 81% of FKBP12.6-/-, but in only 7% of WT mice (P <.05), and were prevented by tetracaine in all FKBP12.6-/- mice. SR Ca(2+) leak in FKBP12.6-/- myocytes was 53% larger than in WT myocytes, and FKBP12.6-/- myocytes showed increased incidence of spontaneous SR Ca(2+) release events, which could be blocked by tetracaine. CONCLUSION: The increased vulnerability to AF in FKBP12.6-/- mice substantiates the notion that defective SR Ca(2+) release caused by abnormal RyR2 and FKBP12.6 interactions may contribute to the initiation or maintenance of atrial fibrillation.

Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy.

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2(R176Q/+) mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2(R176Q/+) mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2(R176Q/+), but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2(R176Q/+) mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2(R176Q/+) mice, but not in controls. Isolated cardiomyocytes from RyR2(R176Q/+) mice exhibited a higher incidence of spontaneous Ca(2+) oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.