Leora B. Balsam

Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium

Under conditions of tissue injury, myocardial replication and regeneration have been reported. A growing number of investigators have implicated adult bone marrow (BM) in this process, suggesting that marrow serves as a reservoir for cardiac precursor cells. It remains unclear which BM cell(s) can contribute to myocardium, and whether they do so by transdifferentiation or cell fusion. Here, we studied the ability of c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1lo Lin- Sca-1+ long-term reconstituting haematopoietic stem cells to regenerate myocardium in an infarct model. Cells were isolated from transgenic mice expressing green fluorescent protein (GFP) and injected directly into ischaemic myocardium of wild-type mice. Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable. These GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45 and myeloid marker Gr-1. We also studied the role of circulating cells in the repair of ischaemic myocardium using GFP+–GFP- parabiotic mice. Again, we found no evidence of myocardial regeneration from blood-borne partner-derived cells. Our data suggest that even in the microenvironment of the injured heart, c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1lo Lin- Sca-1+ long-term reconstituting haematopoietic stem cells adopt only traditional haematopoietic fates.

Novel Index for Invasively Assessing the Coronary Microcirculation

Background—A relatively simple, invasive method for quantitatively assessing the status of the coronary microcirculation independent of the epicardial artery is lacking. Methods and Results—By using a coronary pressure wire and modified software, it is possible to calculate the mean transit time of room-temperature saline injected down a coronary artery. The inverse of the hyperemic mean transit time has been shown to correlate with absolute flow. We hypothesize that distal coronary pressure divided by the inverse of the hyperemic mean transit time provides an index of microcirculatory resistance (IMR) that will correlate with true microcirculatory resistance (TMR), defined as the distal left anterior descending (LAD) pressure divided by hyperemic flow, measured with an external ultrasonic flow probe. A total of 61 measurements were made in 9 Yorkshire swine at baseline and after disruption of the coronary microcirculation, both with and without an epicardial LAD stenosis. The mean IMR (16.9±6.5 U to 25.9±14.4 U, P =0.002) and TMR (0.51±0.14 to 0.79±0.32 mm Hg · mL−1 · min−1, P =0.0001), as well as the % change in IMR (147±66%) and TMR (159±105%, P =NS versus IMR % change), increased significantly and to a similar degree after disruption of the microcirculation. These changes were independent of the status of the epicardial artery. There was a significant correlation between mean IMR and TMR values, as well as between the % change in IMR and % change in TMR. Conclusion—Measuring IMR may provide a simple, quantitative, invasive assessment of the coronary microcirculation.

Insulin‐Like Growth Factor Promotes Engraftment, Differentiation, and Functional Improvement after Transfer of Embryonic Stem Cells for Myocardial Restoration

Insulin‐like growth factor‐1 (IGF‐1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF‐1 might enhance cellular engraftment and host organ‐specific differentiation after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF‐1 to the injected cell population. Green fluorescent protein (GFP)–labeled sv129 ESCs (2.5 × 105) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF‐1 (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF‐1 (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF‐1 group (p = .025). There was a trend toward higher fractional shortening in the IGF‐treated group. Histological analysis demonstrated that IGF‐1 promoted expression of α‐sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF‐1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

Microvascular resistance is not influenced by epicardial coronary artery stenosis severity : experimental validation

Background—The effect of epicardial artery stenosis on myocardial microvascular resistance remains controversial. Recruitable collateral flow, which may affect resistance, was not incorporated into previous measurements. Methods and Results—In an open-chest pig model, distal coronary pressure was measured with a pressure wire, and the apparent minimal microvascular resistance was calculated during peak hyperemia as pressure divided by flow, measured either with a flow probe around the coronary artery (Rmicro app) or with a novel thermodilution technique (apparent index of microcirculatory resistance [IMRapp]). These apparent resistances were compared with the actual Rmicro and IMR after the coronary wedge pressure and collateral flow were incorporated into the calculation. Measurements were made at baseline (no stenosis) and after creation of moderate and severe epicardial artery stenoses. In 6 pigs, 189 measurements of Rmicro and IMR were made under the various epicardial artery conditions. Without consideration of collateral flow, Rmicro app (0.43±0.12 to 0.46±0.10 to 0.51±0.11 mm Hg/mL per minute) and IMRapp (14±4 to 17±7 to 20±10 U) increased progressively and significantly with increasing epicardial artery stenosis (P <0.001 for both). With the incorporation of collateral flow, neither Rmicro nor IMR increased as a result of increasing epicardial artery stenosis. Conclusions—After collateral flow is taken into account, the minimum achievable microvascular resistance is not affected by increasing epicardial artery stenosis.

Comparison of Coronary Thermodilution and Doppler Velocity for Assessing Coronary Flow Reserve

Background—Thermodilution coronary flow reserve (CFRthermo) is a new technique for invasively measuring coronary flow reserve (CFR) with a coronary pressure wire and is based on the ability of the pressure transducer to also measure temperature changes. Whether CFRthermo correlates well enough with absolute flow-derived CFR (CFRflow) to replace Doppler wire–derived CFR (CFRDoppler) remains unclear. Methods and Results—In an open-chest pig model, CFRthermo was measured in the left anterior descending (LAD) artery and compared with CFRDoppler and CFRflow, measured with an external flow probe placed around the LAD. In 9 pigs, CFR was measured simultaneously by all 3 means in the normal LAD and after creation of an epicardial LAD stenosis. To determine the added effect of microvascular disease, measurements of flow reserve were also performed after disruption of the coronary microcirculation with embolized microspheres. Intracoronary papaverine (20 mg) was used to induce hyperemia. In a total of 61 paired measurements, CFRthermo correlated strongly with the reference standard CFRflow (r =0.85, P <0.001). CFRDoppler correlated less well with CFRflow (r =0.72, P <0.001). Bland-Altman analysis showed a closer agreement between CFRthermo and CFRflow. Conclusion—CFRthermo correlates better with CFRflow than does CFRDoppler.

Overexpression of Human Copper/Zinc Superoxide Dismutase (SOD1) Suppresses Ischemia–Reperfusion Injury and Subsequent Development of Graft Coronary Artery Disease in Murine Cardiac Grafts

Background— Ischemia–reperfusion injury is an important risk factor for graft coronary artery disease (GCAD). We hypothesized that overexpression of SOD1 in donor hearts would suppress ischemia—reperfusion injury and thereby reduce GCAD. Methods and Results— In one series, donor hearts of C57BL/6 (H-2 b ) transgenic mice overexpressing human SOD1 or C57BL/6 wild-type mice were heterotopically transplanted into C57BL/6 recipients and procured after 4 hours of reperfusion (n=6 each). Superoxide, TNF-α, and MCP-1/CCL2 production were significantly reduced in the SOD1 transgenic donor heart recipients, and graft injury determined by serum CPK-MB levels was significantly decreased. Cardiomyocyte apoptosis and caspase-3 and caspase-9 activities were significantly decreased in these recipients; caspase-8 activity was unchanged. Fas ligand but not Fas expression was also reduced. In a second series, transgenic and wild-type hearts were transplanted into C-H-2 bm12 KhEg (H-2 bm12 ) recipients, and then procured on day 56 (n=7 each). Cardiac graft beating was significantly better in the SOD1 transgenic donor heart recipients on days 28, 42, and 56 (but not day 14). Significant reduction in luminal narrowing, the intima/media ratio, and the percentage of diseased vessels was seen in the SOD1 transgenic donor heart recipients, and MCP-1/CCL2, ICAM-1, and VCAM-1 production were significantly reduced. Conclusions— Overexpression of SOD1 attenuates both apoptosis and the inflammatory response during ischemia—reperfusion injury and therefore mitigates against the subsequent development of GCAD.

Haematopoietic stem cells and repair of the ischaemic heart

HSCs (haematopoietic stem cells) are multipotent stem cells that give rise to all cells of the blood cell lineage. In recent years, it has been proposed that bone marrow serves as a reservoir for cardiomyogenic precursors and that, following cardiac injury, these stem cells circulate to the site of injury where they contribute to myocardial repair and regeneration. This concept of stem cell plasticity has been controversial and, in fact, several key studies on the cardiomyogenic potential of HSCs have not been reproducible in the hands of independent investigators. Despite this controversy, the clinical community has pushed forward with clinical trials of bone marrow transplantation for the treatment of ischaemic heart disease. The following review summarizes the mechanistic underpinnings of bone marrow transplantation into ischaemic myocardium, focusing on the basic science that forms the foundation of this field, and highlights the controversies and new avenues for research that have emerged. It also describes the current state of the art in clinical trials of bone marrow transplantation for heart failure.

Progression of Alloresponse and Tissue-Specific Immunity during Graft Coronary Artery Disease

Chronic rejection remains the major obstacle for long‐term transplant survival. Both indirect alloresponse and tissue‐specific autoimmunity have been implicated in its pathogenesis. The interrelationship between these two types of host anti‐graft response remains poorly understood. We have developed an immunosuppression‐free mouse model of graft coronary artery disease (GCAD), in which all FVB (H‐2q) cardiac allografts placed into minor Ag (mHC)‐mismatched DBA/1 (H‐2q) hosts survived more than 112 days, and developed GCAD. We then examined the kinetics of both anti‐mHC alloresponse and host autoimmunity against heart‐specific antigen, cardiac myosin (CM). At 8 days post‐transplantation, recipient mice showed minimal intragraft inflammation and apoptosis, and limited expansion of allo‐specific T cells. In addition, we observed early production of anti‐myosin IgG1 autoantibodies, which occurred in the absence of activated CM‐specific T lymphocytes. By day 56, GCAD indices, the numbers of mHC‐ and CM‐reactive T cells, and the levels of circulating allo‐ and CM‐specific antibodies were all significantly increased. While host alloresponse was exhausted at 112 days post‐transplant, T‐cell reactivity against CM persisted. Our data suggest that both allo‐ and tissue‐specific immunity might contribute to the induction of GCAD. They indicate that continual autoimmune response against graft tissue antigens may provide for GCAD sustenance.

Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts.

Background. The oxidative stress associated with ischemia-reperfusion (I/R) of cardiac allografts leads to production of injurious cytokines and expression of proinflammatory adhesion molecules. This is one of the most important alloantigen-independent factors associated with graft coronary artery disease (GCAD). M40401 is a newly developed cell permeable superoxide dismutase mimetic, which has been shown to scavenge superoxide anion with highly specific and enhanced catalytic activity. We hypothesized that M40401 would exert a protective effect in I/R injury of cardiac allografts and ameliorate the progression of GCAD. Methods. Recipient ACI rats were pretreated with M40401 or vehicle control. PVG donor hearts were heterotopically transplanted into the abdomen of ACI recipients. Cardiac allografts were analyzed for adhesion molecule mRNA expression and tumor necrosis factor-&agr; expression after 4 hr of reperfusion. Neutrophil infiltration was detected by myeloperoxidase activity. Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 mRNA were detected by reverse-transcriptase polymerase chain reaction. Immunohistochemical analysis of adhesion molecule expression was also performed. Additional grafts were procured 90 days after transplantation and assessed for the development of GCAD by computer-assisted image analysis. Results. In the M40401-treated group, adhesion molecule expression was significantly less than in the vehicle control group. Treated grafts also had lower myeloperoxidase activity and tumor necrosis factor-&agr; concentration compared with controls. Neointimal proliferation and intima to media ratios in M40401-treated allografts were significantly decreased compared with controls. Conclusions. Selective removal of superoxide anion by M40401 results in inhibition of I/R injury. Furthermore, M40401 treatment decreases the development of oxidative stress-associated GCAD. This treatment strategy may have broad cardioprotective applications for all cardiac operations in addition to cardiac transplantation.

Restoration of blood flow and evaluation of corresponding angiogenic events by scanning electron microscopy after a single dose of VEGF in a model of peripheral vascular disease

The angiogenic effect of vascular endothelial growth factor (VEGF) has typically been assessed by indirect methods, including microsphere injection and angiography. Here, we use 3-D scanning electron microscopy (SEM) to directly visualize patterns of angiogenesis after a single bolus administration of VEGF in a model of peripheral vascular ischemia. Hind limb ischemia was induced by subcutaneous turniquet implantation in adult Wistar rats. The control group (group A, n = 10) was left untreated, group B (n = 10) received a single dose of VEGF (50 μg) injected in the peroneus muscle. LASER Doppler was applied for blood flow measurements. Animals were sacrificed on day 14 after ischemia induction and vascular casting was performed. Angiogenetic events such as ‘tiny lateral sprouts’, ‘arcus formations’, confluences and the angle of sprouting were assessed by SEM. Significant capillary sprouting was observed in both groups. VEGF-treated limbs demonstrated higher degrees of capillary growth (P = 0.01) and flow recovery (P = 0.028). 3-D-SEM showed sprouts to be more frequent in group B. Tiny lateral sprouts, which always left the mother vessel at an angle of 90° and which were of small diameter and lacked imprints of endothelial cell nuclei, were more frequent in the VEGF-treated group (P = 0.018). Arcus formation was significantly higher in the treated group (P = 0.02). We have developed a simple and effective experimental model of ischemia. For the study of angiogenic phenomena, 3-D imaging of the microvasculature offers a direct and conclusive method for the study of angiogenic events.