Jennifer S. Pocius

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction

Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5′-azacytidine, in part depending on Bmpr1a, a receptor for bone morphogenetic proteins. Given intravenously after ischemia/reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre/Lox donor/recipient pair (αMHC-Cre/R26R), differentiation was shown to occur roughly equally, with and without fusion to host cells.

Stem Cell Plasticity in Muscle and Bone Marrow

Abstract: Recent discoveries have demonstrated the extraordinary plasticity of tissue‐derived stem cells, raising fundamental questions about cell lineage relationships and suggesting the potential for novel cell‐based therapies. We have examined this phenomenon in a potential reciprocal relationship between stem cells derived from the skeletal muscle and from the bone marrow. We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5 day in vitro culture were harvested and introduced into each of six lethally irradiated recipients together with distinguishable whole bone marrow cells. Six and twelve weeks later, all recipients showed high‐level engraftment of muscle‐derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56%, indicating that the cultured muscle cells generated approximately 10‐ to 14‐fold more hematopoietic activity than whole bone marrow. Although the identity of the muscle‐derived hematopoietic stem cells is still unknown, they may be identical to muscle satellite cells, some of which lack myogenic regulators and could respond to hematopoietic signals. We have also found that stem cells in the bone marrow can contribute to cardiac muscle repair and neovascularization after ischemic injury. We transplanted highly purified bone marrow stem cells into lethally irradiated mice that subsequently were rendered ischemic by coronary artery occlusion and reperfusion. The engrafted stem cells or their progeny differentiated into cardiomyocytes and endothelial cells and contributed to the formation of functional tissue.

Cardiac muscle plasticity in adult and embryo by heart-derived progenitor cells

Abstract: The evidence of cardiomyocyte proliferation in damaged heart implied cardiac regeneration might occur by resident or extra cardiac stem cells. However, the specification and origin of these cells remain unknown. Here, we report using fluorescence‐activated cell sorting that cardiac progenitor cells resided in adult heart and colocalized with small capillary vessels, within the stem cell antigen (Sca‐1) population expressing high telomerase activity. Notably, hematopoietic stem cells capable of efflux Hoechst 33342, termed side population cells, also were identified within the heart‐derived cells. The cardiac progenitor cells (CD45−/CD34−) express neither cardiac muscle nor endothelial cell markers at an undifferentiated stage. The exposure of 5‐azacytidine induced cardiac differentiation, which depends, in part, on Bmpr1a, a type IA receptor for bone morphogenetic protein (BMP). The capability of adult Sca1+ cells to adopt a cardiac muscle in embryogenesis was substantiated by blastocyst injection, using progenitors from the adult hearts of transgenic mice that harbor a bacterial artificial chromosome expressing GFP via the Nkx‐2.5 locus. Intravenously injected progenitors, shortly after ischemic/reperfusion, homed and functionally differentiated 3.5% of total left ventricle in the host myocardium. Differentiation included both fusion‐independent and fusion‐associated components, proved by the Cre/loxP donor/recipient system. Our studies suggest that endogenous cardiac progenitors reside in the adult heart, regenerate cardiomyocytes functionally, and integrate into the existing heart circuitry.

Cardiomyocyte PDGFR-β signaling is an essential component of the mouse cardiac response to load-induced stress

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

Myocardial infarction and remodeling in mice: effect of reperfusion

Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 ± 1.7% vs. 12 ± 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly ( P < 0.002) increased mass in PO animals (22.5 ± 1.08 mg) vs. OR (17.8 ± 1.10 mg) or sham control (14.8 ± 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.Anatomic and functional changes after either a permanent left anterior descending coronary artery occlusion (PO) or 2 h of occlusion followed by reperfusion (OR) in C57BL/6 mice were examined and compared with those in sham-operated mice. Both interventions generated infarcts comprising 30% of the left ventricle (LV) measured at 24 h and equivalent suppression of LV ejection velocity and filling velocity measured by Doppler ultrasound at 1 wk. Serial follow-up revealed that the ventricular ejection velocity and filling velocity returned to the levels of the sham-operated controls in the OR group at 2 wk and remained there; in contrast, PO animals continued to display suppression of both systolic and diastolic function. In contrast, ejection fractions of PO and OR animals were depressed equivalently (50% from sham-operated controls). Anatomic reconstruction of serial cross sections revealed that the percentage of the LV endocardial area overlying the ventricular scar (expansion ratio) was significantly larger in the PO group vs. the OR group (18 +/- 1.7% vs. 12 +/- 0.9%, P < 0.05). The septum that was never involved in the infarction had a significantly (P < 0.002) increased mass in PO animals (22.5 +/- 1.08 mg) vs. OR (17.8 +/- 1.10 mg) or sham control (14.8 +/- 0.99 mg) animals. Regression analysis demonstrated that the extent of septal hypertrophy correlated with LV expansion ratio. Thus late reperfusion appears to reduce the degree of infarct expansion even under circumstances in which it no longer can alter infarct size. We suggest that reperfusion promoted more effective ventricular repair, less infarct expansion, and significant recovery or preservation of ventricular function.

Pulsed Doppler signal processing for use in mice: applications

We have developed a high-frequency, high-resolution Doppler spectrum analyzer (DSPW) and compared its performance against an adapted clinical Medasonics spectrum analyzer (MSA) and a zero-crossing interval histogram (ZCIH) used previously by us to evaluate cardiovascular physiology in mice. The aortic velocity (means /spl plusmn/ SE: 92.7 /spl plusmn/ 2.5 versus 82.2 /spl plusmn/ 1.8 cm/s) and aortic acceleration (8194 /spl plusmn/ 319 versus 5178 /spl plusmn/ 191 cm/s/sup 2/) determined by the DSPW were significantly higher compared to those by the MSA. Aortic ejection time was shorter (48.3/spl plusmn/ 0.9 versus 64.6 /spl plusmn/ 1.8 ms) and the isovolumic relaxation was longer (17.6 /spl plusmn/ 0.6 versus 13.5 /spl plusmn/0.6 ms) when determined by the DSPW because it generates shorter temporal widths in the velocity spectra when compared to the MSA. These data indicate that the performance of the DSPW in evaluating cardiovascular physiology was better than that of the MSA. There were no significant differences between the aortic pulse wave velocity determined by using the ZCIH (391 /spl plusmn/ 16 cm/s) and the DSPW (394 /spl plusmn/ 20 cm/s). Besides monitoring cardiac function, we have used the DSPW for studying peripheral vascular physiology in normal, transgenic, and surgical models of mice. Several applications such as the detection of high stenotic jet velocities (>4 m/s), vortex shedding frequencies (250 Hz), and subtle changes in wave shapes in peripheral vessels which could not obtained with clinical Doppler systems are now made possible with the DSPW.

Estimating arterial properties from Doppler signals in mice

Mice are the animal of choice for genetic manipulations many of which affect the peripheral vascular system either directly or indirectly via compensatory responses. Traditional pressure and flow methods for assessing vascular function are invasive and difficult to apply to mice so we have been developing several noninvasive techniques using Doppler ultrasound to evaluate the peripheral vasculature in mice. Velocity signals can be obtained from most major peripheral arteries for evaluation of pulsatility and resistance indices, pulse wave velocity can be measured in the aorta and carotid arteries, and the simplified Bernoulli equation can be used to estimate the pressure drop across an aortic stenosis. By measuring velocity simultaneously at two closely spaced sites along an artery, a volume waveform can be generated directly, forward and backward waves can be computed, and pulse pressure can be estimated. Characteristic impedance can be calculated from pulse wave velocity and impedance spectra and reflection coefficients can be calculated. Thus, many of the basic characteristics of the cardiovascular system can be assessed noninvasively in mice using Doppler ultrasound.

Vascular adaptations to transverse aortic banding in mice

Transverse aortic banding in mice generates pressure overload, but cardiac hypertrophy is variable, and the effects on peripheral hemodynamics are unknown. The purpose was to characterize and model carotid and aortic blood flow patterns in banded mice using noninvasive Doppler methods. In 15 normal mice a 27-gauge needle was sutured against the transverse aorta and then removed. In 6 sham-operated mice the suture was not tied. A Doppler probe was used to measure right (R) and left (L) carotid artery (CA), aortic, and mitral blood velocity 1 day later. At 7 days the heart-weight/body-weight ratio (HW/BW) was measured. Mean aortic, mitral, and carotid velocities were similar in sham and banded mice, but peak RCA/LCA velocities were much higher in banded mice and were highly correlated to HW/BW. An esophageal Doppler probe detected high jet velocity and distal vorticity. We conclude that mice compensate for the band by increasing RCA resistance and compliance and decreasing LCA resistance to maintain normal cerebral perfusion. Velocity signals measured within one day and fitted to a lumped-parameter arterial model to estimate the pressure drop can predict the amount of cardiac hypertrophy at one week.