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Repairing cardiac tissue after myocardial infarction (MI) is one of the most challenging goals in tissue engineering. Following ischemic injury, significant matrix remodeling and the formation of avascular scar tissue significantly impairs cell engraftment and survival in the damaged myocardium. This limits the efficacy of cell replacement therapies, demanding strategies that reduce pathological scarring to create a suitable microenvironment for healthy tissue regeneration. Here, we demonstrate the successful fabrication of discrete hyaluronic acid (HA)-based microrods to provide local biochemical and biomechanical signals to reprogram cells and attenuate cardiac fibrosis. HA microrods were produced in a range of physiological stiffness and shown to degrade in the presence of hyaluronidase. Additionally, we show that fibroblasts interact with these microrods in vitro, leading to significant changes in proliferation, collagen expression and other markers of a myofibroblast phenotype. When injected into the myocardium of an adult rat MI model, HA microrods prevented left ventricular wall thinning and improved cardiac function at 6 weeks post infarct.

BackgroundDespite public awareness that tobacco secondhand smoke (SHS) is harmful, many people still assume that marijuana SHS is benign. Debates about whether smoke-free laws should include marijuana are becoming increasingly widespread as marijuana is legalized and the cannabis industry grows. Lack of evidence for marijuana SHS causing acute cardiovascular harm is frequently mistaken for evidence that it is harmless, despite chemical and physical similarity between marijuana and tobacco smoke. We investigated whether brief exposure to marijuana SHS causes acute vascular endothelial dysfunction.Methods and resultsWe measured endothelial function as femoral artery flow-mediated dilation (FMD) in rats before and after exposure to marijuana SHS at levels similar to real-world tobacco SHS conditions. One minute of exposure to marijuana SHS impaired FMD to a comparable extent as impairment from equal concentrations of tobacco SHS, but recovery was considerably slower for marijuana. Exposure to marijuana SHS directly caused cannabinoid-independent vasodilation that subsided within 25 minutes, whereas FMD remained impaired for at least 90 minutes. Impairment occurred even when marijuana lacked cannabinoids and rolling paper was omitted. Endothelium-independent vasodilation by nitroglycerin administration was not impaired. FMD was not impaired by exposure to chamber air.ConclusionsOne minute of exposure to marijuana SHS substantially impairs endothelial function in rats for at least 90 minutes, considerably longer than comparable impairment by tobacco SHS. Impairment of FMD does not require cannabinoids, nicotine, or rolling paper smoke. Our findings in rats suggest that SHS can exert similar adverse cardiovascular effects regardless of whether it is from tobacco or marijuana.

Chronic fibrosis caused by acute myocardial infarction (MI) leads to increased morbidity and mortality due to cardiac dysfunction. We have developed a therapeutic materials strategy that aims to mitigate myocardial fibrosis by utilizing injectable polymeric microstructures to mechanically alter the microenvironment. Polymeric microstructures were fabricated using photolithographic techniques and studied in a three-dimensional culture model of the fibrotic environment and by direct injection into the infarct zone of adult rats. Here, we show dose-dependent down-regulation of expression of genes associated with the mechanical fibrotic response in the presence of microstructures. Injection of this microstructured material into the infarct zone decreased levels of collagen and TGF-β, increased elastin deposition and vascularization in the infarcted region, and improved functional outcomes after six weeks. Our results demonstrate the efficacy of these discrete anti-fibrotic microstructures and suggest a potential therapeutic materials approach for combatting pathologic fibrosis.

IntroductionWe sought to determine the effects of brief exposures to low concentrations of tobacco secondhand smoke (SHS) on arterial flow-mediated dilation (FMD, a nitric oxide-dependent measure of vascular endothelial function), in a controlled animal model never before exposed to smoke. In humans, SHS exposure for 30 min impairs FMD. It is important to gain a better understanding of the acute effects of exposure to SHS at low concentrations and for brief periods of time.MethodsWe measured changes in FMD in rats exposed to a range of real-world levels of SHS for durations of 30 min, 10 min, 1 min, and 4 breaths (roughly 15 s).ResultsWe observed a dose-response relationship between SHS particle concentration over 30 min and post-exposure impairment of FMD, which was linear through the range typically encountered in smoky restaurants and then saturated at higher concentrations. One min of exposure to SHS at moderate concentrations was sufficient to impair FMD.ConclusionsBrief SHS exposure at real-world levels reversibly impairs FMD. Even 1 min of SHS exposure can cause reduction of endothelial function.

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.

Nitric oxide (NO) exerts a wide range of cellular effects in the cardiovascular system. NO is short lived, but S-nitrosoglutathione (GSNO) functions as a stable intracellular bioavailable NO pool. Accordingly, increased levels can facilitate NO-mediated processes, and conversely, catabolism of GSNO by the regulatory enzyme GSNO reductase (GSNOR) can impair these processes. Because dysregulated GSNOR can interfere with processes relevant to cardiovascular health, it follows that inhibition of GSNOR may be beneficial. However, the effect of GSNOR inhibition on vascular activity is unknown. To study the effects of GSNOR inhibition on endothelial function, we treated rats with a small-molecule inhibitor of GSNOR (N6338) that has vasodilatory effects on isolated aortic rings and assessed effects on arterial flow-mediated dilation (FMD), an NO-dependent process. GSNOR inhibition with a single intravenous dose of N6338 preserved FMD (15.3 ± 5.4 vs. 14.2 ± 6.3%, P = nonsignificant) under partial NO synthase inhibition that normally reduces FMD by roughly 50% (14.1 ± 2.9 vs. 7.6 ± 4.4%, P < 0.05). In hypertensive rats, daily oral administration of N6338 for 14 days reduced blood pressure (170.0 ± 5.3/122.7 ± 6.4 vs. 203.8 ± 1.9/143.7 ± 7.5 mmHg for vehicle, P < 0.001) and vascular resistance index (1.5 ± 0.4 vs. 3.2 ± 1.0 mmHg · min · l(-1) for vehicle, P < 0.001), and restored FMD from an initially impaired state (7.4 ± 1.7%, day 0) to a level (13.0 ± 3.1%, day 14, P < 0.001) similar to that observed in normotensive rats. N6338 also reversed the pathological kidney changes exhibited by the hypertensive rats. GSNOR inhibition preserves FMD under conditions of impaired NO production and protects against both microvascular and conduit artery dysfunction in a model of hypertension.

BackgroundEndogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.Methodology/principal findingUsing "middle aged" mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1(+)CD45(-) cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1(+)CD45(-) cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1(+)CD45(-) cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.Conclusions/significanceThese studies demonstrate that cloned Sca-1(+)CD45(-) cells derived from CSs from infarcted "middle aged" hearts are enriched for second heart field (i.e., Isl-1(+)) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.

BackgroundThe extracellular matrix plays an important role in tissue regeneration. We investigated whether extracellular matrix protein fragments could be targeted with antibodies to ischemically injured myocardium to promote angiogenesis and myocardial repair.Methodology/principal findingsFour peptides, 2 derived from fibronectin and 2 derived from Type IV Collagen, were assessed for in vitro and in vivo tendencies for angiogenesis. Three of the four peptides--Hep I, Hep III, RGD--were identified and shown to increase endothelial cell attachment, proliferation, migration and cell activation in vitro. By chemically conjugating these peptides to an anti-myosin heavy chain antibody, the peptides could be administered intravenously and specifically targeted to the site of the myocardial infarction. When administered into Sprague-Dawley rats that underwent ischemia-reperfusion myocardial infarction, these peptides produced statistically significantly higher levels of angiogenesis and arteriogenesis 6 weeks post treatment.Conclusions/significanceWe demonstrated that antibody-targeted ECM-derived peptides alone can be used to sufficiently alter the extracellular matrix microenvironment to induce a dramatic angiogenic response in the myocardial infarct area. Our results indicate a potentially new non-invasive strategy for repairing damaged tissue, as well as a novel tool for investigating in vivo cell biology.

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin E[C.sub.50] = 3.3 x [10.sup.-6] mol/l, peak 21[DELTA]% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine E[C.sub.50] = 1.3 x [10.sup.-6] mol/l, peak 27[DELTA]% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16A% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with [N.sup.G]-monomethyl-L-arginine [1[DELTA]% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16[DELTA]% (SD 3) vs. 10[DELTA]% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats. nitric oxide synthase; endothelium-derived hyperpolarizing factor; rodents; age-dependent endothelial dysfunction

The aim of this study was to determine the feasibility and accuracy of wall motion score index (WMSI) and myocardial performance index (MPI) for measuring regional and global left ventricular (LV) function with use of high-resolution echocardiography after myocardial infarction (MI) in mice. In 48 mice, myocardial infarction was induced by ligation in the middle of the left anterior descending coronary artery. Echocardiography was performed under anesthesia at baseline and 1 mo after MI. WMSI was analyzed by a 16-segment model on short-axis views, and wall motion was scored as 1 for normal, 2 for hypokinetic, 3 for akinetic, 4 for dyskinetic, and 5 for aneurysmal. WMSI was calculated as the sum of scores divided by the total number of segments. MPI was calculated on the basis of isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT), and ejection time (ET): MPI = (IVCT + IVRT)/ET. We measured LV ejection fraction (LVEF), end-systolic and end-diastolic volumes (ESV and EDV), fractional shortening (FS), and infarct size (IS). LVEF at 4 wk after MI was reduced at 32.8 [+ or -] 9.0%. Linear correlation analyses showed that WMSI (1.6 [+ or -] 0.3) correlated with LVEF (r = -0.84, P < 0.0005), FS (r = -0.43, P = 0.003), and IS (34.3 [+ or -] 15.3%, r = 0.86, P < 0.0005). MPI (0.67 [+ or -] 0.09) correlated with LVEF (r = -0.67, P < 0.0005) and IS (r = 0.72, P < 0.0005). MPI also correlated with mitral inflow velocity (r = -0.68, P < 0.0005) and deceleration time (r = -0.42, P = 0.003). Stepwise regression analysis revealed that WMSI was independently associated with IS. IS, FS, mitral inflow velocity, and deceleration time were independent determinants of MPI. In conclusion, echocardiographic assessments of WMSI and MPI in mice are feasible and correlate strongly with two-dimensional measurement of LV function and IS. These novel parameters provide additional noninvasive assessment of regional and global LV function in mice after MI. experimental myocardial infarction; echocardiography; infarct size

The mouse is an important model for the development of therapeutic stem cell/bone marrow cell implantation to treat ischemic myocardium. However, its small heart size hampers accurate implantation into the left ventricular (LV) wall. Precise injections have required surgical visualization of the heart, which is subject to complications and is impractical for delayed or repeated injections. Furthermore, the thickness of the myocardium is comparable to the length of a needle bevel, so surgical exposure does not prevent inadvertent injection into the LV cavity. We describe the use of high-resolution echocardiography to guide nonsurgical injections accurately into the mouse myocardial wall. We optimized this system by using a mixture of ultrasound contrast and fluorescent microspheres injected into the myocardium, which enabled us to interpret the ultrasound image of the needle during injection. Quantitative dye injection studies demonstrated that guided closed-chest injections and open-chest injections deliver comparable amounts of injectate to the myocardium. We successfully used this system in a mouse myocardial infarction model to target the injection of labeled cells to a region adjacent to the infarct. Intentional injection of tracer into the LV cavity resulted in a small accumulation in the myocardium, suggesting that nonguided cell injections into mouse hearts may appear to be successful even if the majority of the injectate is lost in the chamber. The use of this system will allow more precise cellular implantation into the mouse myocardium by accurately guiding injections to desired locations, confirming successful implantation of cells, in a clinically relevant time frame. ultrasound; cell implantation; gene therapy; imaging; stem cells

Concern about the effects of anesthesia on physiological measurements led us to develop methodology to assess left ventricular (LV) pressure in conscious mice. Polyethylene-50 tubing filled with heparinized saline was implanted in the LV cavity through its apex via an abdominal approach and exteriorized to the back of the animal. This surgery was done under anesthesia with either an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg) (K+X) in 11 mice or isoflurane (ISF; 1.5 vol%) by inhalation in 14 mice. Postoperatively, mice were trained daily to lie quietly head first in a plastic cone. LV pressure, the first derivative of LV pressure (dP/dt), and heart rate (HR) in the conscious state were compared between the two groups at 3 days and 1 wk after recovery from surgery using a 1.4-Fr Millar catheter inserted into the LV through the tubing, with the mice lying quietly in the plastic cone. Acutely during anesthesia, K+X decreased HR (from 698 to 298 beats/rain), LV systolic pressure (from 107 to 65 mmHg), and maximal dP/dt (dP/[dt.sub.max]) (from 15,724 to 4,445 mmHg/s), all P < 0.01. Similar but less marked negative chronotropic and inotropic effects were seen with ISF. HR and dP/[dt.sub.max] were decreased significantly in K+X mice 3 days after surgery compared with those anesthetized with ISF (655 vs. 711 beats/min, P < 0.05; 14,448 vs. 18,048 mmHg/s, P < 0.001) but increased to the same level as in ISF mice 1 wk after surgery. In ISF mice, recovery of function occurred rapidly and there were no differences in LV variables between 3 days and 1 wk. LV pressure and dP/dt can be measured in conscious mice with a micromanometer catheter inserted through tubing implanted permanently in the LV apex. Anesthesia with either K+X or, to a lesser extent, ISF, depressed LV function acutely. This depression of function persisted for 3 days after surgery with K+X (but not ISF) and did not recover completely until 1 wk postanesthesia. left ventricular hemodynamics; first derivative of left ventricular pressure; anesthesia; mouse physiology