Human Growth Factor/Immunoglobulin Complexes for Treatment of Myocardial Ischemia-Reperfusion Injury.

Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor 2 (FGF2) are receptor tyrosine kinase agonists that promote cell survival after tissue injury and angiogenesis, cell proliferation and migration during tissue repair and regeneration. Both ligands have potential as systemic treatments for ischemia-reperfusion injury, however clinical use of HGF and FGF2 has been limited by poor pharmacokinetic profiles, i.e., their susceptibility to serum proteases, rapid clearance and short half-lives. Previously, we reported vaso- and cardioprotective protein complexes formed between HGF and polyclonal, non-specific immunoglobulin (IgG) with therapeutic efficacy in a rat model of myocardial ischemia with reperfusion (MI/R). Here, using a pre-clinical porcine MI/R model, we demonstrate human HGF/IgG complexes provide significant myocardial salvage, reduce infarct size, and are detectable in myocardial tissue 24 h after intracoronary injection. Furthermore, we show that multiple daily infusions of HGF/IgG complexes after MI do not lead to production of HGF-specific auto-antibodies, an important concern for administered biologic drugs. In experiments to identify other growth factors that non-covalently interact with IgG, we found that human FGF2 associates with IgG. Similar to human HGF/IgG complexes, FGF2/IgG complexes protected primary human cardiac endothelial cells under simulated ischemia (1% oxygen and nutrient deprivation) for 48-72 h. Molecular modeling studies suggested that FGF2 and HGF both interact with the Fc domain of IgG. Also, we tested whether an Fc-fusion protein would bind FGF2 to form complexes. By native gel electrophoretic assays and biochemical pulldowns, we found that Jagged1, a Notch1 ligand that controls stem cell self-renewal and tissue regeneration, bound FGF2 when presented as a Jagged1- Fc fusion protein. Our results suggest that human growth factor/IgG and FGF2/Fc- fusion complexes have potential to provide a biologics platform to treat myocardial ischemia-reperfusion and other forms of tissue injury.

A Cyber-Physical System for Near Real-Time Monitoring of At-Home Orthopedic Rehabilitation and Mobile-Based Provider-Patient Communications to Improve Adherence: Development and Formative Evaluation.

BACKGROUND: Knee extensor muscle performance is reduced after lower extremity trauma and orthopedic surgical interventions. At-home use of neuromuscular electrical stimulation (NMES) may improve functional recovery, but adherence to at-home interventions is low. Greater benefits from NMES may be realized with closer monitoring of adherence to at-home prescriptions and more frequent patient-provider interactions. OBJECTIVE: This study aimed to develop a cyber-physical system to monitor at-home adherence to NMES prescription and facilitate patient-provider communications to improve adherence in near real time. METHODS: The RehabTracker cyber-physical system was developed to accomplish this goal and comprises four components: (1) hardware modifications to a commercially available NMES therapy device to monitor device use and provide Bluetooth functionality; (2) an iPhone Operating System-based mobile health (mHealth) app that enables patient-provider communications in near real time; (3) a clinician portal to allow oversight of patient adherence with device use; and (4) a back-end server to store data, enable adherence analysis, and send automated push notifications to the patient. These four elements were designed to be fully compliant with the Health Insurance Portability and Accountability Act. The system underwent formative testing in a cohort of patients following anterior cruciate ligament rupture (n=7) to begin to assess face validity. RESULTS: Compared with the NMES device software-tracked device use, the RehabTracker system recorded 83% (40/48) of the rehabilitation sessions, with 100% (32/32) of all sessions logged by the system in 4 out of 7 patients. In patients for whom tracking of automated push notifications was enabled, 100% (29/29) of the push notifications sent by the back-end server were received by the patient. Process, hardware, and software issues contributing to these inaccuracies are detailed. CONCLUSIONS: RehabTracker represents a promising mHealth app for tracking and improving adherence with at-home NMES rehabilitation programs and warrants further refinement and testing.

High dose intracoronary N-acetylcysteine in a porcine model of ST-elevation myocardial infarction.

We sought to evaluate the safety and efficacy of N-acetylcysteine (NAC) on ischemia and reperfusion in a pig model focusing on cardio-renal protection. High doses of NAC may provide protection from contrast induced nephropathy (CIN). NAC has also been demonstrated to reduce myocardial infarction size and improve left ventricular function after ischemia in both humans and animals studies. In this study we tested the safety and cardiorenal protective efficacy of intracoronary NAC delivered in the radiographic contrast agent in a pig model that simulates the catheter based reperfusion therapy of ST elevation myocardial infarctions. 27 pigs underwent 45 min of ischemia after surgical ligation of distal left descending coronary artery. With coronary reperfusion the animals received at total of 200 mL of the contrast agent Iopamidol with and without NAC to mimic radiographic contrast use during invasive reperfusion therapy. At 24 h the following endpoints were compared: LV function (MRI, echocardiography), myocardial injury (infarct size, area-at-risk, troponin, creatinine kinase) and CIN (creatinine, BUN and renal histology). The effects of NAC on platelet reactivity were also evaluated. Intracoronary administration of NAC administered in the contrast agent is safe. NAC reduces platelet reactivity and there was a trend towards a better cardiac function at 24 h. There was no significant difference in the size of the myocardial infarction. In this model of ischemia-reperfusion high dose NAC did not protect from CIN. High dose intracoronary NAC administered with the radiographic contrast is safe but does not provide significant cardio-renal protection.

Intracellular distributions of essential elements in cardiomyocytes.

We describe the intracellular distributions of nine essential elements (P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn) found in cardiomyocytes imaged using synchrotron X-ray induced fluorescence. Cardiomyocytes were isolated from rat hearts, flash frozen on Si(3)N(4) windows, freeze-dried, and imaged with approximately 300 nm spatial resolution. Distinct longitudinal patterns in cardiomyocytes were most apparent for the elements Fe and Cu, which clearly colocalized. Transverse striations were apparent for P, S, Fe, and Zn, while those for Zn were consistently the most prominent ( approximately 10(-3)M) and appeared with a periodicity in the range 1.63-1.75 microm, the expected length of a sarcomere. Transverse striations for high concentrations of P, Fe, and Zn and low concentrations of S colocalized and coincided with the I-band of the intact cardiomyocyte. Fluorescence microscopy using FluoZin-3 in intact cardiomyocytes suggests that Zn(2+) influx is through sarcolemmal calcium channels and that significant stores of intracellular Zn(2+) may be released quickly (<1s) into the cytosol. These data collectively suggest that Zn(2+) is buffered by structures associated near the T-tubules and/or in the sarcoplasmic reticulum and is found in relative abundance sufficient to act as a modifier of Ca(2+) regulation or as a possible signaling messenger for gene expression.

Cardiomyocyte function associated with hyperactivity and/or hypertension in genetic models of LV hypertrophy.

We examined cardiomyocyte intracellular calcium ([Ca2+]i) dynamics and sarcomere shortening dynamics in genetic rat models of left ventricular (LV) hypertrophy associated with or without hypertension (HT) and with or without hyperactive (HA) behavior. Previous selective breeding of the spontaneously hypertensive rat (SHR) strain, which is HA and HT, with the Wistar-Kyoto (WKY) rat strain, which is not hyperactive (NA) and not hypertensive (NT), has led to two unique strains: the WKHA strain, selected for HA and NT, and the WKHT strain, selected for NA and HT. Cardiomyocytes were isolated from young adult males and females of each strain, paced at 2, 3, and 4 Hz in 1.2 mM external Ca2+ concentration at 37 degrees C, and cardiomyocyte [Ca2+]i and sarcomere dynamics were recorded simultaneously. Under these conditions, LV cardiomyocyte systolic and diastolic [Ca2+]i dynamics and diastolic sarcomere dynamics in the WKHT were significantly enhanced compared with WKY controls, suggesting an underlying LV hypertrophic response that successfully compensated for HT in the absence of HA. LV cardiomyocyte [Ca2+]i dynamics in the WKHA and SHR were strikingly similar to each other and only slightly reduced compared with WKY. LV cardiomyocyte systolic and diastolic sarcomere dynamics, on the other hand, were significantly reduced in the SHR compare with WKHA and more so in male than in female SHR. We conclude from these data that HT alone is an insufficient descriptor of the cause of LV hypertrophy and diminished LV cardiomyocyte function in the SHR rat. These data further suggest that HA (augmented by male sex) in the SHR may interact with the HT state to initiate impaired cardiomyocyte function and thereby inhibit or undermine an otherwise compensatory response that may occur with HT in the absence of HA.