Ray C.-J. Chiu

Cellular cardiomyoplasty: Myocardial regeneration with satellite cell implantation

BACKGROUND Damaged skeletal muscle is able to regenerate because of the presence of satellite cells, which are undifferentiated myoblasts. In contrast, destruction of cardiac myocytes is associated with an irreversible loss of myocardium and replacement with scar tissue, because it lacks stem cells. We tested the hypothesis that skeletal muscle satellite cells implanted into injured myocardium can differentiate into cardiac muscle fibers and thus repair damaged heart muscle. METHODS Two series of canine studies were performed. In the first series (n = 26), satellite cells were isolated from skeletal muscle, cultured, and labeled with tritiated thymidine. The cells were implanted into acutely cryoinjured myocardium and the specimens harvested 4 to 18 weeks later. In the second series (n = 20), satellite cells in culture were labeled with lacZ reporter gene, which encodes production of Escherichia coli beta-galactosidase. Four to 6 weeks later, beta-galactosidase activity was studied using X-Gal stain. RESULTS New striated muscles were found in the first series of experiments at the site of implantation, within a dense scar created by cryoinjury. These muscles showed histologic evidence of intercalated discs and centrally located nuclei, similar to those seen in cardiac muscle fibers. Tritiated thymidine radioactivity was not identified clearly, presumably due to dilutional effect as the stem cells replicated repeatedly. In the second series, histochemical studies of reporter gene-labeled and implanted satellite cells revealed the presence of beta-galactosidase within the cells at the implant site, which confirmed the survival of implanted cells. CONCLUSIONS Our data are consistent with the hypothesis of milieu-influenced differentiation of satellite cells into cardiac-like muscle cells. Confirmation of these findings and its functional capabilities could have important clinical implications.

Xenotransplant cardiac chimera: immune tolerance of adult stem cells

BACKGROUND Bone marrow stromal cells have been shown to engraft into xenogeneic fetal recipients. In view of the potential clinical utility as an alternative source for cellular and gene therapies, we studied the fate of xenogeneic marrow stromal cells after their systemic transplantation into fully immunocompetent adult recipients without immunosuppression. METHODS Bone marrow stromal cells were isolated from C57B1/6 mice and retrovirally transduced with LacZ reporter gene for cell labeling. We then injected 6 x 10(6) labeled cells into immunocompetent adult Lewis rats. One week later, the recipient animals underwent coronary artery ligation and were sacrificed at various time points ranging from 1 day to 12 weeks after ligation. Hearts, blood, and bone marrow samples were collected for histologic and immunohistochemical studies. RESULTS Labeled mice cells engrafted into the bone marrow cavities of the recipient rats for at least 13 weeks after transplantation without any immunosuppression. On the other hand, circulating mice cells were positive only for the animals with 1-day-old myocardial infarction. At various time points, numerous mice cells could be found in the infarcted myocardium that were not seen before coronary ligation. Some of these cells subsequently showed positive staining for cardiomyocyte specific proteins, while other labeled cells participated in angiogenesis in the infarcted area. CONCLUSIONS The marrow stromal cells are adult stem cells with unique immunologic tolerance allowing their engraftment into a xenogeneic environment, while preserving their ability to be recruited to an injured myocardium by way of the bloodstream and to undergo differentiation to form a stable cardiac chimera.

Cell transplantation for myocardial repair: an experimental approach.

Myocardium lacks the ability to regenerate following injury. This is in contrast to skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of satellite cells. It was hypothesized that SKM satellite cells multiplied in vitro could be used to repair injured heart muscle. Fourteen dogs underwent explantation of the anterior tibialis muscle. Satellite cells were multiplied in vitro and their nuclei were labelled with tritiated thymidine 24 h prior to implantation. The same dogs were then subjected successfully to a myocardial injury by the application of a cryoprobe. The cells were suspended in serum-free growth medium and autotransplanted within the damaged muscle. Medium without cells was injected into an adjacent site to serve as a control. Endpoints comprised histology using standard stains as well as Masson trichrome (specific for connective tissue), and radioautography. In five dogs, satellite cell isolation, culture, and implantation were technically satisfactory. In three implanted dogs, specimens were taken within 6-8 wk. There were persistence of the implantation channels in the experimental sites when compared to the controls. Macroscopically, muscle tissue completely surrounded by scar tissue could be seen. Masson trichrome staining showed homogeneous scar in the control site, but not in the test site where a patch of muscle fibres containing intercalated discs (characteristic of myocardial tissue) was observed. In two other dogs, specimens were taken at 14 wk postimplantation. Muscle tissue could not be found. These preliminary results could be consistent with the hypothesis that SKM satellite cells can form neo-myocardium within an appropriate environment. Our specimens failed to demonstrate the presence of myocyte nuclei. It is therefore further hypothesized that in the late postoperative period, the muscle regenerate failed to survive.

Angiogenesis and growth factor expression in a model of transmyocardial revascularization.

BACKGROUND The mechanism by which transmyocardial revascularization (TMR) exerts a beneficial effect remains unknown. We hypothesize that the myocardial punctures of TMR cause a myocardial injury, leading to an angiogenic response mediated by a number of growth factors. METHODS Fifty-three rats underwent ligation of the left coronary artery. Group I (n = 25) served as controls, whereas group II (n = 28) underwent concomitant TMR by the creation of six transmural channels with a 25-gauge needle in the ischemic zone. Surviving animals in both groups were sacrificed at intervals of 1, 2, 4, and 8 weeks (n = 5 in each subgroup). Immunohistochemistry in the infarct areas was performed for factor VIII to assess vascular density. Immunohistochemistry using specific antibodies was also performed for transforming growth factor-beta, basic-fibroblast growth factor, and vasoendothelial growth factor. Growth factor expression was quantitated by comparing areas of staining (in mm2) with computerized morphometric analysis. RESULTS Mortality was similar in both groups (5/25 versus 8/28; not significant). Group II had significantly greater vascular density than group I (5.65 versus 4.06 vessels/high-power field; p < 0.001), with a peak at 1 week postoperatively (9.12 versus 5.56 vessels/high-power field; p < 0.0001) in both groups. Overall, levels of both transforming growth factor-beta and basic-fibroblast growth factor were significantly higher in the TMR group compared with the control group (0.207 versus 0.141 mm2/mm2, p < 0.05; and 0.125 versus 0.099 mm2/ mm2, p < 0.05). CONCLUSIONS This model of TMR is associated with a significant angiogenic response, which appears to be mediated by the release of certain angiogenic growth factors such as transforming growth factor-beta and basic-fibroblast growth factor. With the long-term patency of laser-created myocardial channels in clinical TMR increasingly in doubt, its mechanism of myocardial revascularization may be similar to that observed in our model.

The effects of prosthetic cardiac binding and adynamic cardiomyoplasty in a model of dilated cardiomyopathy

OBJECTIVE Because adynamic cardiomyoplasty, or wrapping skeletal muscle around the heart, had been shown to provide a girdling effect and delay progressive ventricular dilatation in heart failure, a similar girdling effect by the much simpler procedure of cardiac binding, using a prosthetic membrane to wrap the heart, was studied and compared with that of adynamic cardiomyoplasty. METHODS Twenty-one dogs were divided into control, adynamic cardiomyoplasty, and cardiac binding groups. Cardiac dimension and hemodynamic studies were carried out before and 4 weeks after rapid pacing at 250 beats/min. For adynamic cardiomyoplasty, the left latissimus dorsi muscle was used for the cardiac wrap; for cardiac binding, a Marlex sheet (C. R. Bard, Inc., Murray Hill, N.J.) was used. Serial two-dimensional echocardiography, right heart catheterization, and in the cardiac binding group, left heart catheterization were performed. RESULTS Four weeks of rapid pacing induced severe heart failure and cardiac dilatation. The magnitude of ventricular dilatation at the end of rapid pacing was less in the cardiac binding group than in the control group and least in the adynamic cardiomyoplasty group. Left ventricular end-diastolic volume, end-systolic volume, and ejection fraction were 82.1 +/- 21.1 ml, 67.1 +/- 16.0 ml, and 17.5% +/- 5.8%, respectively, in the control group; 61.9. +/- 8.1 ml, 44.1 +/- 7.8 ml, and 30.1% +/- 3.6%, respectively, in the cardiac binding group; and 51.8 +/- 8.7 ml, 30.3 +/- 10.4 ml, and 27.0% +/- 4.0%, respectively, in the adynamic cardiomyoplasty group. CONCLUSIONS Both adynamic cardiomyoplasty and cardiac binding reduced cardiac enlargement and functional deterioration after rapid pacing, with adynamic cardiomyoplasty appearing to be more effective, perhaps because of the adaptive capabilities of the skeletal muscle wrap. However, cardiac binding is a simpler and less invasive procedure, which may be useful as an adjunct to prevent or delay progressive ventricular dilatation in heart failure.

Percutaneous endoscopic tracheostomy

Bedside percutaneous tracheostomies are increasingly performed. This avoids patient transport to the operating room. Complications of this procedure are largely related to the blind nature of the technique. After laboratory studies, 4 patients underwent percutaneous endoscopic guided tracheostomy in a selective clinical trial. There were no procedure-related complications. Endoscopic guidance ensures precise low tracheostomy position, prevents paratracheal tube misplacement, and avoids inadvertent injuries.

Concise Review: Immunomodulatory Properties of Mesenchymal Stem Cells in Cellular Transplantation: Update, Controversies, and Unknowns

Stem cell transplantation is a promising approach for improving cardiac function after severe myocardial damage, for which the use of autologous donor cells has been preferred to avoid immune rejection. Recently, however, rodent as well as human mesenchymal stem cells have been reported to be uniquely immune‐tolerant, in both in vitro and in vivo transplant models. In this review, we explore in detail the current understanding of the underlying immunologic mechanisms, which can facilitate the use of such cells as “universal donor cells” with fascinating clinical implications.

Angiogenesis in transmyocardial revascularization: comparison of laser versus mechanical punctures

BACKGROUND Transmyocardial laser revascularization (TMLR), which has been shown to reduce angina in clinical trials, was originally based on the belief that laser channels are unique and can remain patent. An increasing body of evidence indicates otherwise, and transmyocardial revascularization (TMR) angiogenesis is currently thought to be induced by nonspecific inflammatory response to tissue injuries. We tested the hypothesis that mechanical transmyocardial revascularization (TMMR) may induce angiogenic responses similar to that seen with lasers. METHODS Ameroid constrictors were implanted around proximal circumflex arteries of porcine hearts. Six weeks later, they were randomly assigned (n = 5 each) to receive 10 transmural channels in the ischemic zone by a carbon dioxide laser (group I) or by a needle (group II). A third group (group III) had 30 needle channels in the same area, while a control group (group IV) received no TMR. The hearts were harvested 1 week later, and, using immunohistochemistry, vascular endothelial growth factor (VEGF) expression was studied and quantified by computerized morphometric analysis. Densities of vascular structures positively stained for VEGF per high-power field (HPF) were also compared. RESULTS Virtually no TMR channels remained patent histologically. Group III had a significant higher level of total VEGF expression (14.18+/-0.78 mm2) compared with group I (7.07+/-2.06 mm2, p < 0.001) and group II (4.74+/-3.35 mm2, p < 0.001). Vascular density was significantly elevated in all treatment groups compared with the control (group I, 7.7+/-0.8/HPF vs group II, 4.5+/-2.3/HPF vs group III, 8.1+/-0.6/HPF vs group IV, 1.1+/-0.5/HPF). CONCLUSIONS In view of the significant cost implications, our findings that needle punctures may also induce angiogenic response comparable with that with laser suggest that it is justifiable and desirable to include a TMMR arm for comparison with TMLR in future clinical trials.

Microencapsulation to reduce mechanical loss of microspheres: implications in myocardial cell therapy §

OBJECTIVE Previous regenerative studies have demonstrated massive cell losses after intramyocardial cellular delivery. Therefore, efforts at reducing mechanical losses may prove more successful in optimising cellular therapy. In this study, we hypothesized that escalating mesenchymal stem cells (MSCs) dose will not produce corresponding improvement in cardiac function due to washout of the small cells in microcirculation. Using microspheres similar in size to MSCs, that are encapsulated in alginate-poly-l-lysine-alginate (APA), we tested the hypothesis that size is an important factor in early losses. METHODS In experiment I, five groups of rats (n=9 each) underwent coronary ligation; group I had no treatment; the other groups received escalating 0.5 × 10(6), 1.5 × 10(6), 3 × 10(6) and 5 × 10(6) of MSCs each. Echocardiogram was performed at baseline, 2 days and 7 weeks after surgery. In experiment II, cell-sized microspheres (10 μm) were encapsulated in APA microcapsules. In group I (n=16), rats received bare microspheres, group II (n=16) microspheres within 200 μm microcapsules and in group III (n=16), microspheres within 400 μm microcapsules. After 20 min, hearts were quantified for the amount retained. RESULTS Myocardial function did not improve further with escalating cell doses beyond an initial response at 1.5 × 10(6) cells. Encapsulated microspheres in 200 μm and 400 μm microcapsules demonstrated a fourfold increase in retention rate compared with 10 μm microspheres. CONCLUSION We concluded that suboptimal functional improvement in this animal model starts at 1.5 × 10(6) cells and does not respond to escalating cell doses. Improving mechanical retention is possible by increasing the size of the injectate. Microencapsulation could be used to encapsulate donor cells and facilitate functional improvement in cellular heart failure therapy.

Angiogenic response induced by mechanical transmyocardial revascularization.

BACKGROUND Angiogenesis is the proposed mechanism of transmyocardial revascularization. We evaluated mechanical transmyocardial revascularization in a chronically ischemic porcine model by measuring myocardial angiogenic response. METHODS Ameroid constrictors were implanted 6 weeks before mechanical transmyocardial revascularization. Group I (n = 5) and group II (n = 3) animals received 30 punctures with an 18-gauge needle and samples were harvested at 1 and 4 weeks, respectively, after the operation. Group III (n = 5) had sternotomy only and served as the control group. Myocardial samples were immunohistochemically stained for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-beta) using specific antibodies. Growth factor expression was quantified by means of computer-assisted morphometry. Vascular density was assessed by immunohistochemical stain for VEGF and factor VIII. RESULTS Compared with group III, increased angiogenic factor levels were found in group I (VEGF 0.47 +/- 0.03 mm(2) vs 0.05 +/- 0.05 mm(2), P =.000; bFGF 0.67 +/- 0.14 mm(2) vs 0.03 +/- 0.03 mm(2), P =. 000; TGF-beta 1.40 +/- 0.18 mm(2) vs 0.09 +/- 0.06 mm(2), P = 0.000), and in group II (VEGF 0.34 +/- 0.06 mm(2) vs 0.05 +/- 0.05 mm(2), P =.003; bFGF 0.06 +/- 0.02 mm(2) vs 0.03 +/- 0.03 mm(2), P =.135; TGF-beta 0.28 +/- 0.09 mm(2) vs 0.09 +/- 0.06 mm(2), P =.042). Vascular densities after mechanical transmyocardial revascularization were also increased (group I, VEGF stain 8.1 +/- 0. 6 vs 1.1 +/- 0.5, P =.000; factor VIII stain 5.1 +/- 2.7 vs 0.4 +/- 0.3, P =.018; group II, VEGF stain 1.9 +/- 0.5 vs 1.1 +/- 0.5, P = 0. 107; factor VIII stain 2.3 +/- 0.4 vs 0.4 +/- 0.3, P =.004). CONCLUSIONS Mechanical transmyocardial revascularization is associated with increased angiogenic factor expression and concomitant neovascularization at up to 4 weeks. These changes are indistinguishable from those of laser transmyocardial revascularization. Myocardial perfusion studies are needed to establish the functional significance of these angiogenic changes.