Anguo Gu

Angiogenesis is enhanced in ischemic canine myocardium by transmyocardial laser revascularization

OBJECTIVES This study sought to test whether transmyocardial laser revascularization (TMLR) stimulates angiogenesis in an animal model of chronic ischemia. BACKGROUND TMLR relieves angina and may also improve blood flow in patients who are not candidates for traditional therapies. The mechanisms of these benefits are not fully defined. METHODS Ischemia was created in 14 dogs by proximal left anterior descending coronary ameroid constrictors. TMLR was performed in the anterior wall (approximately 1 channel/cm2) of seven dogs; the remaining dogs served as the ischemic control group. Myocardial blood flow was measured (colored microspheres) at rest and during chemical stress (adenosine) in the acute setting and after 2 months. RESULTS TMLR did not influence blood flow in the acute setting. After 2 months, resting blood flow increased comparably in the anterior wall in both groups to approximately 80% of normal. However, the TMLR-treated dogs demonstrated an approximately 40% increase in blood flow capacity during stress in the ischemic territory compared with untreated dogs (left anterior descending coronary artery/left circumflex coronary artery flow 0.53+/-0.16 in the control group vs. 0.73+/-0.08 in TMLR animals, p < 0.05). Vascular proliferation, assessed by bromodeoxyuridine incorporation and proliferating cell nuclear antigen positivity in endothelial and smooth muscle cells was about four times greater in the TMLR group than in the control group (p < 0.001). The density of vessels with at least one smooth muscle cell layer was approximately 1.4 times greater in the myocardium surrounding the TMLR channel remnants than in control ischemic tissue (p < 0.001). CONCLUSIONS In this canine model of chronic ischemia, TMLR significantly enhances angiogenesis as evidenced by the increased number of vessels lined with smooth muscle cells, markedly increased vascular proliferation and increased blood flow capacity during stress.

Does blood flow through holmium: YAG transmyocardial laser channels?

BACKGROUND Early reports indicate that transmyocardial laser revascularization improves symptoms in patients with refractory angina. However, there is little experimental evidence of whether blood flow through channels is the mechanism of action. METHODS Endocardial channels were made in the distribution of the left anterior descending coronary artery in canine hearts (n = 5) using a holmium:yttrium-aluminum garnet laser. Hearts were excised acutely while perfused in a retrograde fashion from a second dog so that the aortic valve always remained closed. The proximal left anterior descending coronary artery was ligated. To measure direct transmyocardial blood flow, colored microspheres were injected into the left ventricular chamber. RESULTS The number of spheres per gram of tissue in the channel region was significantly higher than in the control region (low load, 302.5 +/- 169.0 versus 41.8 +/- 59.4; high load, 208.4 +/- 138.3 versus 5.8 +/- 11.7; both, p < 0.05). However, the estimated regional blood flow through the channels was extremely low (<0.01 mL/g/min. In the chronic setting (n = 4) (2-week survival), no flow as detected through the channels, and the endocardial entry points were closed. CONCLUSIONS Transmyocardial blood flow does not appear to occur through channels made with the holmium:yttrium-aluminum garnet laser. It remains to be determined whether this is the case with other types of lasers.

Physiology, histology, and 2-week morphology of acute transmyocardial channels made with a CO2 laser

BACKGROUND Transmyocardial revascularization with a CO2 laser appears to improve symptoms in patients with refractory angina. However, it remains controversial as to whether blood flow through the channels is the mechanism of benefit, especially in the acute setting. METHODS AND RESULTS Three protocols were used to test whether blood flows through transmyocardial CO2 laser revascularization channels. First, channels were made in excised, cross-perfused dog hearts (n = 5) using a CO2 laser (The Heart Laser; PLC Systems Inc, Milford, MA; 40 J/pulse) followed by ligation of the proximal left anterior descending coronary artery. Colored microspheres injected into the left ventricular chamber failed to detect any significant transmyocardial blood flow. In the second protocol (n = 4), laser channels were created in the left anterior descending artery territory, the left anterior descending artery was ligated, and the hearts were excised after 24 hours. Triphenyltetrazolium chloride staining revealed that no viable myocardium was detected around the laser channels in the ischemic myocardium. Finally, channels examined 2 weeks after creation in normal (n = 6) or ischemic (n = 4) myocardium did not maintain their original caliber but were invaded by granulation tissue, which included a large amount of smaller vascular spaces and vessels of various sizes. CONCLUSIONS Transmyocardial laser revascularization channels made with this CO2 laser did not provide acute myocardial perfusion or preserve myocardial viability in the face of acute ischemia. Channel morphology changes dramatically within the first 2 weeks. To the degree that these findings pertain to human myocardium, the results suggest that transmyocardial blood flow may not be the mechanism of benefit of this procedure, particularly in the acute setting.

β2-Adrenergic Stimulation Attenuates Left Ventricular Remodeling, Decreases Apoptosis, and Improves Calcium Homeostasis in a Rodent Model of Ischemic Cardiomyopathy

The benefit of the β2-adrenergic agonist, clenbuterol, in left ventricular assist device patients with dilated cardiomyopathy has been reported, but its effect on ischemic heart failure (HF) is unknown. We investigated whether clenbuterol improves left ventricular remodeling, myocardial apoptosis and has synergy with a β1 antagonist, metoprolol, in a model of ischemic HF. Rats were randomized to: 1) HF only; 2) HF + clenbuterol; 3) HF + metoprolol; 4) HF + clenbuterol + metoprolol; and 5) rats with sham surgery. HF was induced by left anterior descending artery (LAD) artery ligation and confirmed by decreased left ventricular fractional shortening, decreased maximum left ventricular dP/dt (dP/dtmax), and elevated left ventricular end-diastolic pressure (LVEDP) compared with sham rats (p < 0.01). After 9 weeks of oral therapy, echocardiographic, hemodynamic, and ex vivo end-diastolic pressure-volume relationship (EDPVR) measurements were obtained. Immunohistochemistry was performed for myocardial apoptosis and DNA damage markers. Levels of calcium-handling proteins were assessed by Western blot analysis. Clenbuterol-treated HF rats had increased weight gain and heart weights versus HF rats (p < 0.05). EDPVR curves revealed a leftward shift in clenbuterol rats versus metoprolol and HF rats (p < 0.05). The metoprolol-treated group had a lower LVEDP and higher dP/dtmax versus the HF group (p < 0.05). Clenbuterol and metoprolol groups had decreased myocardial apoptosis and DNA damage markers and increased DNA repair markers versus HF rats (all p < 0.01). Protein levels of the ryanodine receptor and sarcoplasmic reticulum calcium-ATPase were improved in clenbuterol-, metoprolol-, and clenbuterol+metoprolol-treated groups versus HF rats. However, as a combination therapy, there were no synergistic effects of clenbuterol+metoprolol treatment. We conclude that clenbuterol ameliorates EDPVR, apoptosis, and calcium homeostasis but does not have synergy with metoprolol in our model of ischemic HF.

BLOOD FLOW CAPACITY VIA DIRECT ACUTE MYOCARDIAL REVASCULARIZATION

The concept of direct myocardial revascularization, achieving myocardial perfusion through means other than the normal coronary vasculature, has a long history with the most widely investigated technique being the Vineberg procedure; current interest centers around the encouraging preliminary clinical results obtained with transmyocardial laser revascularization. Despite significant previous research, the acute blood flow potential through the direct myocardial route remains unknown. Nontransmural laser channels were made in the distal LAD territory from the epicardial surface of 5 mongrel dogs to which an internal mammary artery was connected. A flow probe was placed on the distal most portion of the artery and an intercostal branch was cannulated for infusion of colored microspheres. Measurements were taken under baselin conditions and following LAD and epicardial collateral ligation. Under all conditions, blood flow pattern was of a to-and-fronature. At baseline, there was an average 0.60±0.24 ml/min net flow into the myocardium which was all contained within 0.5 cm of the central channel with a final myocardial perfusion of 0.011±0.016 ml/min/g. Following induction of ischemia average flow increased to 1.41±0.51 ml/min which extended as far as 1 cm from the channel with a final myocardial perfusion of 0.22±0.19 ml/min/g. In conclusion, a limited amount of acute myocardial perfusion can be achieved by the present technique of direct myocardial revascularization and the amount of flow is highly dependent upon the amount of flow through the native circulation.

Radio frequency transmyocardial revascularization enhances angiogenesis and causes myocardial denervation in canine model.

Transmyocardial revascularization (TMR) relieves angina and improves exercise tolerance in patients. Angiogenesis and myocardial denervation have been proposed as factors contributing to these benefits. To test whether radio frequency transmyocardial revascularization (RF‐TMR) enhances angiogenesis and causes myocardial denervation.

Bradycardic Therapy Improves Left Ventricular Function and Remodeling in Dogs with Coronary Embolization-Induced Chronic Heart Failure

Both β-adrenergic blockade and bradycardia may contribute to the therapeutic effect of β-blockers in chronic heart failure (CHF). This study tested the relative importance of bradycardia by comparing cilobradine (Cilo), a sinus node inhibitor, with a β-blocker, metoprolol (Meto), in an established canine model of CHF. Dogs were chronically instrumented for hemodynamic and left ventricular (LV) volume measurements. CHF was created by daily coronary embolization via a chronically implanted coronary (left anterior descending coronary artery) catheter. After establishment of CHF, control (n = 6), Meto (30 mg/day, n = 5), Cilo (low) (1 mg/kg/day, n = 5), or Cilo (high) (3 mg/kg/day, n = 5) was given orally for 12 weeks. Systemic hemodynamics, echocardiography, and pressure volume analysis were measured at baseline, at CHF, and 3 months after treatment in an awake state. Protein levels of cardiac sarcoplasmic reticulum calcium-ATPase (SERCA2a), ryanodine receptor (RyR2), and Na+-Ca2+ exchanger (NCX1) were measured by Western blot. RyR2 protein kinase A (PKA) phosphorylation was determined by back-phosphorylation. After 12 weeks, Meto and Cilo (high and low) produced similar bradycardic effects, accompanied by a significantly improved LV dP/dt versus control [Meto, 2602 ± 70; Cilo (low), 2517 ± 45; Cilo (high), 2579 ± 78; control, 1922 ± 115 mm Hg/s; p < 0.05]. Both Meto and Cilo (high) normalized protein levels of SERCA2a and NCX1 and reversed PKA hyperphosphorylation of RyR2, in contrast to controls. High-dose cilobradine effectively produced bradycardia and improved cardiac function after CHF, comparable with metoprolol. Restored protein levels of SERCA2a and improved function of RyR2 may be important mechanisms associated with cilobradine therapy.

Functional consequences of acute collagen degradation studied in crystalloid perfused rat hearts

Objectives: The impact of acute collagen disruption by the disulfide donor, 5,5′-dithio-2-nitrobenzoic acid (DTNB) on ventricular properties was tested in rat hearts.Methods: Collagen was degraded acutely in 13 isolated, isovlumically contracting rat hearts by perfusion with 1 mM DTNB added to Krebs-Henseleit solution for 1 hour followed by 2-hour perfusion with normal solution. Another 13 hearts were perfused with normal solution for 3 hours (Control).Results: Collagen content was 3.5±0.5% of ventricular dry weight in control group compared with 2.1±0.4% in DTNB group (decrease by 40%, p<0.01). Scanning electron micrographs revealed loss of the delicate collagen network surrounding muscle fibers in DTNB treated hearts. Developed pressure at a fixed volume decreased to 86±17% of the baseline value after 3-hour perfusion in the control group, whereas in DTNB treated hearts developed pressure fell to 68±13% (p<0.01). End-diastolic pressure was set at 5 mmHg at the beginning of the experiment and rose to 15±8 mmHg in control and 30±13 mmHg (p<0.01) in the treated hearts. Concomitantly, wet-to-dry weight ratio increased from 5.63±0.26 in control to 6.07±0.11 (p<0.05) in the DTNB treated hearts. A separate set of experiments on isolated myocytes excluded the possibility of a direct effect of DTNB on myocyte contractile function.Conclusions: These data suggested that with 40% collagen disruption by DTNB there is a significant increase in tissue edema that results in a decrease in chamber capacitance; in addition, there is a significant decrease in systolic performance which reflects the combined effect of edema and loss of collagen.

Macrophage inhibitor, Semapimod, reduces tumor necrosis factor-alpha in myocardium in a rat model of ischemic heart failure

Pharmaceutical agents aimed at reducing tumor necrosis factor-alpha (TNF-α) levels appeared to be attractive possibilities in the medical management of heart failure, as heart failure was shown to be associated with high TNF-α levels. However, therapies specifically targeting TNF-α failed to show any survival benefit. We examined whether a broad inhibition of inflammatory cytokine production secondary to macrophage inhibition would be more effective at improving cardiac function in the setting of heart failure. To this end, we studied Semapimod (formerly known as CNI-1493), a synthetic guanylhydrazone that inhibits macrophage activation and the production of several inflammatory cytokines. Left anterior descending coronary ligation surgery was performed on each animal to induce a myocardial infarction. After confirming heart failure by echocardiography, the animals were randomly assigned to one of four groups: (1) rats with myocardial infarct receiving high-dose Semapimod, 10 mg/kg/d (MI-H, N = 13); (2) rats with myocardial infarct receiving low-dose Semapimod, 3 mg/kg/d (MI-L, N = 9); (3) rats with myocardial infarct receiving vehicle treatment, 2.5% mannitol in water (MI-0, N = 9); and (4) control rats with sham operation and vehicle treatment (Sham, N = 10). Both Semapimod and vehicle treatments were administered by daily tail vein injections over a course of five days. Echoes were repeated at 2, 5, and 9 weeks following treatment. At 9 weeks, hemodynamic data were collected and the animals were euthanized. Trichrome staining was done to assess infarct, and immunohistochemistry was performed to assess TNF-α levels. Prior to drug administration, serum was taken from 5 random rats. No detectable level of TNF-α was seen (lower limit of detection for the assay used = 12.5 pg/mL). Also prior to any treatment, echocardiography confirmed significant cardiac impairment of rats undergoing LAD ligation as compared with sham. Over the course of the 9 weeks, there were 4 deaths, all in the MI-H group. There was no difference between Semapimod-treated animals and vehicle-treated MI animals in any echocardiographic or hemodynamic parameter. TNF-α staining in the noninfarcted region was evident only in the MI groups, not the sham group. When blindly compared on a semiquantitative scale (ie, 0 = no visible staining to 3 = marked staining), a significant difference in staining was observed between MI-0 versus MI-H (1.19 ± 0.32 versus 0.33 ± 0.14; P = 0.03) and between MI-0 and MI-L (1.19 ± 0.32 versus 0.39 ± 0.22; P = 0.05). In this setting, despite the fact that Semapimod treatment decreased tissue TNF-α levels, it did not improve cardiac function, and at high doses it was associated with higher mortality. These results in a rodent model confirm the results of clinical trials with etanercept and infliximab (ie, that decreasing TNF levels in plasma or tissues does not improve cardiac function and may actually increase mortality).

Factors contributing to pressure overload-induced immediate early gene expression in adult rat hearts in vivo.

We determined the contributions of angiotensin II type 1 receptor (AT1) stimulation, adrenergic stimulation, and autonomic activation to pressure overload-induced c- fos expression in the adult rat heart in vivo. c- fos expression was increased in pressure-overloaded hearts created by aortic banding compared with sham-operated rats (458 ± 100% vs. sham, P < 0.05). GR-138950, a selective AT1 antagonist, did not blunt this expression (banding vs. banding + GR-138950: 458 ± 100% vs. 500 ± 125%, not significant). Atropine and hexamethonium partially decreased c- fos expression (banding vs. banding + atropine/hexamethonium: 700 ± 67% vs. 400 ± 67%, P < 0.05). Phentolamine had no significant effect on c- fosexpression; however, propranolol inhibited the expression (banding vs. banding + propranolol: 492 ± 108% vs. 154 ± 15%, P < 0.05). The inhibition by propranolol was independent of the decreases in heart rate. Thus factors contributing to pressure overload-induced c- fos expression in adult rat hearts in vivo are different from those in neonatal myocytes in vitro undergoing stretch.We determined the contributions of angiotensin II type 1 receptor (AT(1)) stimulation, adrenergic stimulation, and autonomic activation to pressure overload-induced c-fos expression in the adult rat heart in vivo. c-fos expression was increased in pressure-overloaded hearts created by aortic banding compared with sham-operated rats (458 +/- 100% vs. sham, P < 0.05). GR-138950, a selective AT(1) antagonist, did not blunt this expression (banding vs. banding + GR-138950: 458 +/- 100% vs. 500 +/- 125%, not significant). Atropine and hexamethonium partially decreased c-fos expression (banding vs. banding + atropine/hexamethonium: 700 +/- 67% vs. 400 +/- 67%, P < 0.05). Phentolamine had no significant effect on c-fos expression; however, propranolol inhibited the expression (banding vs. banding + propranolol: 492 +/- 108% vs. 154 +/- 15%, P < 0.05). The inhibition by propranolol was independent of the decreases in heart rate. Thus factors contributing to pressure overload-induced c-fos expression in adult rat hearts in vivo are different from those in neonatal myocytes in vitro undergoing stretch.