Mitral valve (MV) repair is the preferred method of correcting mitral valve regurgitation. Realistic analysis of MV function and prediction of successful repair should include both the left ventricle (LV) and the MV to capture their coupled behavior. However, simultaneous imaging and processing of LV and MV has been a challenge. We present a novel and efficient method to convert transesophageal echocardiography (TEE) images into virtual 3D models for enhanced visualization and predictive flow modeling to assist with presurgical planning. A total of 5 pigs were anesthetized prior to obtaining 3D TEE. Resulting DICOM data was processed using Slicer software. MV anatomy while maximally opened was segmented using its Valve Segmentation tool. LV anatomy at the start and end of diastole was segmented in Slicer. The MV was smoothened and converted into a shell in MeshLab then unified with the LV using Meshmixer. 3D models generated with our workflow were validated against the harvested MV and LV tissue. The generated 3D model of both MV and LV at end diastole geometrically reasonable correlation with the harvested sample. Unified anatomically accurate virtual models of the MV-LV for each animal throughout diastole were successfully developed. Virtually, the annular circumference, LV length from annulus to apex, posterior MV leaflet, anterolateral leaflet, and anteromedial leaflet were 11.8, 5.1, 1.9, 1.8, and 1.7 cm. Anatomically, the corresponding measurements are 8.6, 6.0, 1.4, 1.0, and 1.1 cm. In conclusion, we have developed an efficient, relatively automated workflow to create 3D models of MVs and LVs for hemodynamic modeling and visualization. This algorithm and 3D protocol may help to improve our mitral valve understanding prior to planning surgical repair.

Background: Xe-liposome therapy has shown cerebroprotection in adult stroke animals. This study aims to validate the Xe therapeutic effect by MRI in adult rats and the therapeutic effect in an aged animal. Method: A 2-hour MCAO was induced in adult rodents. The MCAO animals were then divided into two groups: one receiving no treatment and the other receiving Xe-liposomal treatment (3 doses within 2-6 hours after stroke followed by 1 dose per day). Diffusion-weighted MRI conducted at 2, 6, and 24 hours post-MCAO, were analyzed by our image center. For aged rats (14 rats, 74 weeks old, male and female), MCAO (1.5 hours) was induced and then divided into two groups: stroke without treatment (n=5) and stroke with Xe-liposome treatment (n=9) and given 3 doses at day one and once per day for following days. Neurological function was tested by Limb placement and rotarod, and infarct volumes were measured two days post-stroke/treatment. Results: MRI (Fig.) demonstrated that infarct volume growth ratio was lower in the Xe-treated animals than those without treatment, indicating a protective effect of Xe against ischemic injury. MCAO induced 25% ± 5.4% infarct volume in the aged animals. Xe- reduced the infarct size to 6.7% ± 3.4%. Xe treatment demonstrated improved neurological functional recovery in these aged stroke animals. Conclusions: Xe-liposomal treatment reduces ischemic core growth in adult rats and has a therapeutic effect in aged stroke animals. These results support further development of Xe-based therapies for stroke.

Liposomes as carriers for CRISPR/Cas9 complexes represent an attractive approach for cardiovascular gene therapy. A critical barrier to this approach remains the efficient delivery of CRISPR-based genetic materials into cardiomyocytes. Echogenic liposomes (ELIP) containing a fluorescein isothiocyanate-labeled decoy oligodeoxynucleotide against nuclear factor kappa B (ELIP-NF-κB-FITC) were used both in vitro on mouse neonatal ventricular myocytes and in vivo on rat hearts to assess gene delivery efficacy with or without ultrasound. In vitro analysis was then repeated with ELIP containing Cas9-sg-IL1RL1 (interleukin 1 receptor-like 1) RNA to determine the efficiency of gene knockdown. ELIP-NF-κB-FITC without ultrasound showed limited gene delivery in vitro and in vivo, but ultrasound combined with ELIP notably improved penetration into heart cells and tissues. When ELIP was used to deliver Cas9-sg-IL1RL1 RNA, gene editing was successful and enhanced by ultrasound. This innovative approach shows promise for heart disease gene therapy using CRISPR technology.

Peri-stent restenosis following stent implantation is a major clinical problem. We have previously demonstrated that ultrasound-facilitated liposomal delivery of pioglitazone (PGN) to the arterial wall attenuated in-stent restenosis. To evaluate ultrasound mediated arterial delivery, in Yucatan miniswine, balloon inflations were performed in the carotid and subclavian arteries to simulate stent implantation and induce fibrin formation. The fibrin-binding peptide, GPRPPGGGC, was conjugated to echogenic liposomes (ELIP) containing dinitrophenyl-L-alanine-labelled pioglitazone (DNP-PGN) for targeting purposes. After pre-treating the arteries with nitroglycerine, fibrin-binding peptide-conjugated PGN-loaded ELIP (PAFb-DNP-PGN-ELIP also termed atheroglitatide) were delivered to the injured arteries via an endovascular catheter with an ultrasound core, either with or without ultrasound application (EKOS (TM) Endovascular System, Boston Scientific). In arteries treated with atheroglitatide, there was substantial delivery of PGN into the superficial layers (5 mu m from the lumen) of the arteries with and without ultrasound, [(1951.17 relative fluorescence units (RFU) vs. 1901.17 RFU; P-value = 0.939)]. With ultrasound activation there was increased penetration of PGN into the deeper arterial layers (up to 35 mu m from the lumen) [(13195.25 RFU vs. 7681.00 RFU; P-value = 0.005)]. These pre-clinical data demonstrate ultrasound mediated therapeutic vascular delivery to deeper layers of the injured arterial wall. This model has the potential to reduce peri-stent restenosis.

Atherosclerosis is a complex, multi-stage disease characterized by pathological changes across the vascular wall. Endothelial dysfunction, inflammation, hypoxia, and vascular smooth muscle cell proliferation contribute to its progression. An effective strategy capable of delivering pleiotropic treatment to the vascular wall is essential to limit neointimal formation. Echogenic liposomes (ELIP), which can encapsulate bioactive gases and therapeutic agents, have the potential to deliver enhanced penetration and treatment efficacy for atherosclerosis. In this study, liposomes loaded with nitric oxide (NO) and rosiglitazone, a peroxisome proliferator-activated receptor agonist, were prepared using hydration, sonication, freeze-thawing, and pressurization. The efficacy of this delivery system was evaluated in a rabbit model of acute arterial injury induced by balloon injury to the common carotid artery. Intra-arterial administration of rosiglitazone/NO co-encapsulated liposomes (R/NO-ELIP) immediately following injury resulted in reduced intimal thickening after 14 days. The anti-inflammatory and anti-proliferative effects of the co-delivery system were investigated. These liposomes were echogenic, enabling ultrasound imaging to assess their distribution and delivery. R/NO-ELIP delivery exhibited a greater attenuation (88 ± 15%) of intimal proliferation when compared to NO-ELIP (75 ± 13%) or R-ELIP (51 ± 6%) delivery alone. The study demonstrates the potential of echogenic liposomes as a promising platform for ultrasound imaging and therapeutic delivery.

We have conducted a stability study of a complex liposomal pharmaceutical product, Atheroglitatide (AGT), stored at three temperatures, 4, 24, and 37 °C, for up to six months. The six parameters measured were functions of liposomal integrity (size and number), drug payload (loading efficiency), targeting peptide integrity (conjugation efficiency and specific avidity), and echogenicity (ultrasound-dependent controlled drug release), which were considered most relevant to the product’s intended use. At 4 °C, liposome diameter trended upward, indicative of aggregation, while liposome number per mg lipid and echogenicity trended downward. At 24 °C, peptide conjugation efficiency (CE) and targeting efficiency (TE, specific avidity) trended downward. At 37 °C, CE and drug (pioglitazone) loading efficiency trended downward. At 4 °C, the intended storage temperature, echogenicity, and liposome size reached their practical tolerance limits at 6 months, fixing the product expiration at that point. Arrhenius analysis of targeting peptide CE and drug loading efficiency decay at the higher temperatures indicated complete stability of these characteristics at 4 °C. The results of this study underscore the storage stability challenges presented by complex nanopharmaceutical formulations.

Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment.

To demonstrate thrombolytic efficacy of a tissue plasminogen activator (tPA)-loaded echogenic liposome (TELIP) formulation in a rabbit thrombotic stroke model (the most relevant animal model for evaluation of directed thrombolytic therapy for ischemic stroke), we sought to develop a means of monitoring thrombus dissolution quantitatively by ultrasound imaging methods. We hypothesized that a gas-free ultrasound contrast agent can be incorporated into blood clots at a concentration that does not affect the tPA-mediated clot dissolution rate, while enabling quantitative assessment of the clot dissolution rate. Clots were formed from a mixture of whole rabbit blood, 1 M calcium chloride, human thrombin and varying amounts of microcrystalline cellulose. Washed clots in tubes were weighed at 30, 60 and 90 minutes after addition of recombinant tPA (rtPA) in porcine plasma (100 μg/ml). Clot echogenicity at each time point was assessed using a Philips HDI 5000 ultrasound system using an L12-5 linear array probe. Recorded Images underwent videodensitometric analysis that converted image reflectivity to mean gray scale values (MGSV). We found that 1.12 mg/ml of microcrystalline cellulose in rabbit blood clots (0.2 ml) provided optimal echogenicity without affecting clot dissolution rates (0.3-0.6 mg/min.) caused by rtPA. The clot dissolution rate measured by videodensitometric analysis of the echogenic clots agreed well with that determined by mass loss measurements (0.28% 0-time value/minute). This method will be important for demonstrating in vivo efficacy with potentially decreased hemorrhagic effects provided by directed tPA vehicles relative to systemic administration of the free thrombolytic.

Acute ischemic stroke induces widespread cellular necrosis and apoptosis and activation of inflammation. These mediate release of cell-free DNA (cfDNA) into the circulation. Although increased cfDNA concentrations have been associated with clinical outcome in stroke, data concerning neuronal cfDNA is rare. In this study, we assessed the association of cfDNA with neuron biomarkers in patients with acute ischemic stroke. Methods: Thirteen patients without stroke and four patients with middle cerebral artery occlusion were recruited at UT Memorial Hermann Hospital. Blood plasma samples were collected and cfDNA samples were prepared for whole-genome bisulfite sequencing (WGBS) in order to generate quantitative DNA methylation datasets. The resulting WGBS data were analyzed for epigenetic biomarker identification using bioinformatic statistical approaches. CelFiE was used for cell deconvolution, designed to accurately estimate the relative abundances of cell types and tissues present in plasma cfDNA from methylation sequencing, a technique frequently referred to as cell deconvolution. Results: Differential methylation statistical analysis comparing the stroke against the non-stroke group of samples found 3493 DNA methylation differences. Pathway Analysis discovered greater enrichment for neuronal function with the top hit being “Neuronal System” followed by activity surrounding synapses, demonstrating that a clear enrichment of methylation change in neuronal pathways can be reflected from blood plasma samples following the stroke. Cell deconvolution showed trends of increased megakaryocyte and neutrophils which are involved in the post-stroke immune response. It may be that the secreted neutrophil nuclear DNA in blood plasma drives the increase in the observed proportion of neutrophil cell type in the stroke group samples. Principal Component Analysis demonstrated good separation between the stroke and normal groups. Conclusion: Blood sample can be used for circulating cfDNA analysis and neuron biomarker discovery for acute ischemic stroke prediction/recovery.

Introduction: Ischemic stroke is one of the main causes of long-term morbidity/mortality and early treatment is key. Xenon (Xe) has demonstrated powerful neuroprotective effects on both ischemic and hemorrhagic stroke, but its clinical and long-term outpatient administration is limited by currently available delivery methods. Development of an oral Xe formulation is an attractive strategy for stroke treatment in the field. We developed a Xenon (Xe)-cyclodextrin (CD) oral formulation. Hypothesis: Xe-CD formulation can be delivered orally for acute stroke treatment. Methods: A stable, oral Xe-CD clathrate (Xe solid gas) was formed by pressurizing Xe (3 atm) into 14% alpha-cyclodextrin at room temperature. Xe concentration was measured by GC-MS. Rat middle cerebral artery occlusion (MCAO) was induced by intraluminar suture. The animals were divided into groups: stroke (n=4); stroke with CD (n=3); stroke with 0.5 ml Xe-CD (n=3); stroke with 1.0 ml Xe-CD (n=3); and stroke with 1.5 ml Xe-CD (n=3). Treatments were administered by gavage once a day for 3 days. At day 3, neurological behavior testing was conducted. The infarct size and neuronal death were assessed and normalized by total brain volume. Results: There are 1.56 mM Xe clathrate per 0.6 mM alpha-cyclodextrin. MCAO for 2 hours induced 21% ± 3.6% infarct volume. Xe-CD treatment dose-dependently reduced the infarct size to 18.9% ± 2.3% by 0.5 ml Xe-CD, 6.7% ± 1.8% by 1.0 ml Xe-CD, and 4.8% ± 1.2% by 1.5 ml Xe-CD (Fig. 1A). Behavioral test assessment using forelimb placement rate and Rotarod score matched infarct size. TUNEL staining demonstrated significant decreases in apoptosis in stroke rats treated with 1.5 ml Xe-CD (Fig. 1B). Conclusions: We have demonstrated oral Xe-CD formulation ameliorates neuronal apoptosis and reduces infarct size. Xe-CD represents a promising therapeutic for ischemic stroke treatment.

Introduction: Genome editor-CRISPR/Cas9 holds much promise for treatment of heart disease. A critical step is the development of gene transfer platform that can deliver genes into heart muscle efficiently. We have developed ultrasound (US)-responsive echogenic liposomes (ELIP) for controlled payload release, and enhanced payload penetration upon ultrasound application. Hypothesis: We hypothesize that ELIP in combination with ultrasound application can enhance gene delivery in cardiomyocytes. Methods: FITC (green fluorescence) labeled NF-kB oligonucleotides were used as a prototype for gene delivery. ELIP containing NF-kB-FITC (ELIP-NF-kB-FITC) were prepared by a conventional procedure of hydrating the lipid film, sonication, freezing and drying. Rats were divided into control, ELIP-NF-kB-FITC without US, and ELIP-NF-kB-FITC with US. ELIP-NF-kB-FITC were administered into the left ventricle by injection with the aorta clamped. Ultrasound (1 MHz at 0.5 W/cm2, 100% duty cycle) was applied to the heart for 5 min. Thereafter, heart was excise, fixed and imaged by fluorescent microscopy. The fluorescent intensity of the left ventricular wall was quantified by NIH ImageJ software. Results: ELIP-NF-kB-FITC delivered without ultrasound application showed limited green fluorescence in the heart. Ultrasound (US) in combination with ELIP enhanced NF-kB-FITC penetration into the ventricular walls (Fig. 1A). Quantitatively, ultrasound provided 2-fold increase (2.0 ± 0.27, P < 0.01 vs control) in the mean green fluorescence (Fig. 1 B). Conclusions: Ultrasound application in combination with ELIP enhanced gene delivery into the ventricular walls. This platform offers a promising delivery system of CRISPR components for the correction of cardiomyopathies caused by gene mutation/change.

Introduction: Epigenetic markers including 5-metylcytosine (5mC) can reflect disease severity and treatment effect. Stroke is a multifactorial disease causing aberrant DNA methylation (DNAm) and memory damage. Hypothesis: Models built for 5mC biomarker panel signatures may be useful for quantitative prediction of post-stroke long-term memory recovery in response to treatment. Methods: Stroke was induced with a middle cerebral artery occlusion (MCAO)(18 rats: sham(6), stroke(6), and stroke with treatment (6)). Xe-liposomes were intravenously administrated repetitively for 3 days after MCAO. At day 35, memory was assessed and scaled using a novel objective recognition test. Brain hippocampus and cortex tissue samples were prepared for whole-genome bisulfite sequencing (WGBS) to generate quantitative DNA methylation datasets in response to repetitive intermittent Xe administration. The resulting WGBS data were evaluated and quantitatively associated with memory recovery. Results: A post-stroke long-term memory outcome model was generated with scaled data using the following inputs: (1) good recovery, (2) moderate recovery, (3) edge, (4) light damage, and (5) severe damage. Differential DNAm 5mC analysis was performed comparing MCAO affected hemisphere with sham and Xe-treated samples. Initial 5mC differential analysis identified 6,175 (Cortex), and 9,037 (Hippocampus) CpG sites that were different between groups. Subsequently the LASSO algorithm was applied and selected 22 CpG sites that correlated with the long-term memory recovery scale. PCA analysis using the 22 CpGs demonstrated separation of memory outcomes. We have also built a predictive linear regression model of the 22 CpGs to assess memory loss or recovery in response to treatment. Analysis showed 7 out of 22 CpGs are conserved in humans (orthologs) for 30 bp regions. Conclusions: Epigenetic 5mC marker panels have the potential for quantitating post-stroke memory damages/recovery.

Late in-stent restenosis remains a significant problem. Bare-metal stents were implanted into peripheral arteries in miniature swine, followed by direct intra-arterial infusion of nitric oxide-loaded echogenic Liposomes (ELIPs) and anti-intercellular adhesion molecule-1 conjugated ELIPs loaded with pioglitazone exposed to an endovascular catheter with an ultrasonic core. Ultrasound-facilitated delivery of ELIP formulations into stented peripheral arteries attenuated neointimal growth. Local atheroma-targeted, ultrasound-triggered delivery of nitric oxide and pioglitazone, an anti-inflammatory peroxisome proliferator-activated receptor-gamma agonist, into stented arteries has the potential to stabilize stent-induced neointimal growth and obviate the need for long-term antiptatetet therapy. (C) 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

Background: Epigenetic markers represent a useful and reliable biomarker of pathogenesis of diseases. Stroke is a multifactorial disease causing aberrant DNA methylation profiles. We have found tha...

Xenon (Xe) is a bioactive gas capable of reducing and stabilizing neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for xenon loading and ultrasound-triggered release. Microbubbles loaded with either xenon (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) (9:1 v/v) were synthesized by high-shear mixing. The size distribution and the frequency-dependent attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband acoustic attenuation spectroscopy system, respectively. The Xe dose was evaluated using gas chromatography/mass spectrometry. The total Xe doses in Xe-MB and Xe-OFP-MB were 113.1 ± 13.5 and 145.6 ± 25.5 μl per mg of lipid, respectively. Co-encapsulation of OFP increased the total xenon dose, attenuation coefficient, microbubble stability (in an undersaturated solution), and shelf life of the agent. Triggered release of gas payload was demonstrated with 6-MHz duplex Doppler and 220-kHz pulsed ultrasound. These results constitute the first step toward the use of lipid-shelled microbubbles for applications such as neuroprotection in stroke.

Cardiac hypertrophy often causes impairment of cardiac function. Xenon (Xe), a naturally occurring noble gas, is known to provide neurological and myocardial protection without side effects. The conventional method of Xe delivery by inhalation is not feasible on a chronic basis. We have developed an orally deliverable, effective Xe formulation for long-term administration. We employed 2-hydroxypropyl)-β-cyclodextrin (HPCD), which was dissolved in water to increase the Xe concentration in solution. The beneficial effects of long-term oral administration of Xe-enriched solutions on cardiovascular function were evaluated in vivo. HPCD increased Xe solubility from 0.22 mM to 0.67 mM (3.8-fold). Aged ApoE knockout mice fed high-fat diet for 6 weeks developed hypertension, and myocardial hypertrophy with impaired cardiac function. Oral Xe prevented this ischemic damage, preserving normal blood pressure, while maintaining normal left ventricular mass and wall thickness. This novel formulation allows for gastrointestinal delivery and cardiovascular stabilization.