SDF-1α Preserves Cardiac Function After Infarction
To test the hypothesis that SDF-1α protein could improve cardiac function post-infarction, we created ligations of the left anterior descending coronary artery in adult male mice to prevent blood flow to a portion of the left ventricle, creating a zone of injury. Injections of PBS or SDF-1α were administered into myocardium at two sites near the infarct zone. Mice were then subjected to echocardiography at various time points to measure cardiac function by assessment of fractional shortening (FS) and ejection fraction (EF). All studies and analyses of data were performed in blinded fashion.
We created myocardial infarctions in adult male mice and treated half with SDF-1α and half with PBS. At 14 days post-infarction, left ventricles of PBS-treated mice had a mean FS of 27.9 +/– 1.5% (n=9). SDF-1α treatment resulted in a mean FS of 38.1 +/– 1.5% (n=11; P < .0001). As a second measure of ventricular function, two-dimensional echocardiographic measurements revealed that the mean fraction of blood ejected from the left ventricle (EF) in PBS-treated mice was 35.0 +/– 7.9%, compared to a mean of 61.9 +/– 3.7% (P < .0001) in SDF-1α-treated mice. (). At 28 days after infarction, when additional ventricular remodeling has occurred and the scar is typically well formed, we observed a similar trend in cardiac function of SDF-1α-treated mice. FS was 26.8 +/– 1.2% (n=9) for the PBS group and 39.2 +/– 2.9% (n=11; P < .0001) for the SDF-1α group, while EF was 31.5 +/– 3.5% and 48.8 +/– 2.4% (P < .0001) for PBS and SDF-1α groups, respectively (). Cardiac function remained depressed relative to sham-operated animals (~60% FS; ~75% EF). The improvement at 28 days in FS or EF (46% and 55%, respectively) upon SDF-1α treatment corresponded to echocardiographic findings that the end diastolic dimensions (EDD) and end systolic dimensions (ESD) were both significantly smaller in the SDF-1α group, indicating that SDF-1α treatment had resulted in increased cardiac function and decreased cardiac dilation after infarction (). Finally, we found that SDF-1α administration in the absence of infarction did not lead to an increase in cardiac function (data not shown).
Figure 1 SDF-1α treatment after coronary ligation improves myocardial function in vivo. (A) Distribution of left ventricular fractional shortening at 1, 3, 14, and 28 days after coronary ligation with or without SDF-1α treatment. Means and 95% (more ...)
Histological analysis revealed a marked reduction in the size of the scar tissue area and therefore a thicker functional anterior wall of the heart upon SDF-1α treatment (). By 6 weeks post-infarction, the ratio of scar tissue circumferential length to left ventricle circumferential length in SDF-1α-treated animals was reduced by 56% from that seen in PBS-treated controls (P < .001). At 9 weeks post-infarction, the reduction of scar circumference in SDF-1α-treated hearts was 43% relative to controls (P < .001; ). The functional improvement persisted in these animals corresponding to the scar improvement.
Figure 2 SDF-1α reduces levels of scar tissue post-infarction. Representative trichrome staining of transverse heart sections 42 days after coronary ligation and PBS (A, B) or SDF-1α (C, D) treatment. Collagen in scar is indicated in blue. Higher (more ...)
The functional and histologic improvements observed with the single administration of SDF-1α immediately after coronary ligation suggested that the beneficial effects of SDF-1α may occur in the early stages following infarction. We therefore sought to determine the timeframe of functional improvement by performing echocardiography at numerous time points within days of the coronary ligation. Remarkably, as early as 1 day after infarction, we found that FS was 32.2 +/– 1.6% (n=8) with PBS treatment compared to 40.2 +/– 1.6% (n=8, P < 0.0001) with SDF-1α treatment; correspondingly, EF was 40.7 +/– 2.7% (n=8) or 56.6 +/– 3.7% (n=8, P < 0.0001), respectively. This pattern continued 3 days post-infarction as SDF-1α treated mice again demonstrated significant improvement in FS and EF ().
SDF-1α-mediated functional improvement occurred as early as 24 hours post-infarction and continued 3, 14, and 28 days post-infarction. We performed parallel experiments with thymosin β4 to investigate the comparative efficacy of SDF-1α and found that improvement of cardiac function after coronary ligation was similar with SDF-1α or thymosin β4. Interestingly, the combination of SDF-1α and thymosin β4 appeared to have no greater effect than either one alone, suggesting a lack of synergy (Supp. Figure 1
). One potential explanation for this observation is that the beneficial effects may occur through similar downstream pathways or mechanisms that are already maximized.
SDF-1α Promotes Survival of Ischemic Myocardium
Our previous data with thymosin β4 suggest that it functions in a cardioprotective fashion within 24 hours after infarction, possibly followed by neoangiogenesis, rather than through recruitment or promotion of stem cell differentiation into cardiomyocytes. However, there are reports suggesting that SDF-1α can attract CXCR4-expressing hematopoietic stem cells to the heart, where they are assumed to take up residence and improve cardiac function22, 23
. The mechanism by which the stem cells might improve function remains unclear. Whether stem cells differentiate into functional cardiomyocytes has been controversial, but recent studies have suggested that secreted signals arising from stem cells may somehow potentiate cardiac regeneration or repair24-26
To investigate the mechanism by which SDF-1α induces cardiac repair, we examined the degree of cell death in the direct area of infarction and the neighboring area of hypoxic myocardium. Apoptotic cells marked by TUNEL assay were observed in both control and SDF-1α-treated hearts during the first 24 hours, and were largely isolated to the border zone located immediately adjacent to the area of infarct (). However, by 72 hours post-infarction, the apoptosis had spread outside of the immediate area of infarction to surrounding myocardial tissue in all directions in the control PBS-treated hearts. In contrast, the SDF-1α-treated hearts showed little or no apoptosis outside of the area of infarct (). Remote myocardium in both groups remained free of any significant apoptosis at these early time points. Co-staining with muscle actin confirmed that cells undergoing apoptosis were indeed myocytes (). Quantification revealed that at 24 hours post-infarction, 84.0 +/– 19.6 or 60.7 +/– 11.8 apoptotic myocytes per field of view were present in the immediately adjacent border myocardium of PBS- or SDF-1α-treated hearts, respectively; however, this difference was not statistically significant. But by 72 hours, a highly significant difference was observed with 63.3 +/– 16.4 or 1.3 +/– 1.5 apoptotic myocytes per field of view in the border myocardium of PBS-or SDF-1α-treated hearts, respectively (P < .0001; ). Thus, bordering myocardium that is normally irreparably damaged post-infarction is protected by SDF-1α-directed cell survival.
Figure 3 SDF-1α promotes cell survival post-infarction. TUNEL-positive cells (bright green) in bordering myocardium adjacent to the zone of infarct 24 hours after coronary ligation and PBS (A, B) or SDF-1 (C, D) treatment. TUNEL-positive cells in bordering (more ...)
SDF-1α Treatment After Myocardial Infarction Results in Increased Angiogenesis
While the cardioprotective effects of SDF-1α may aid in survival of hypoxic myocardium, the myocytes ultimately would need to be vascularized to achieve long-term survival. Hence, we investigated the degree of neo-angiogenesis in the presence of SDF-1α. An antibody to isolectin B4, a known marker of endothelial cells in the microvasculature, demonstrated a significant increase in the number of capillaries in the area of injury (border zone) in SDF-1α-treated hearts compared to PBS-treated hearts within 72 hours (). Quantification of the isolectin B4-positive capillaries revealed an approximately 93% increase in microvasculature over controls (). This observation was validated with two other endothelial markers, PECAM and vWF (data not shown). Increased capillary density was not observed in unaffected regions of the myocardium. It is difficult to establish, however, if the increased amount of vasculature bordering the area of infarction is a direct effect of SDF-1α administration, an indirect effect of greater initial cell survival leading to recruitment of new vasculature, or a combination of the two.
Figure 4 SDF-1α treatment induces increased angiogenesis 72 hours post-infarction. Immunohistochemistry using isolectin B4 antibody staining to mark endothelial cells demonstrated a higher density of microvasculature in bordering myocardium close to the (more ...)
SDF-1α Treatment Increases Akt Phosphorylation and Levels of VEGF Protein
Our previous observations of ILK and Akt activation upon thymosin β4 treatment as well as SDF-1α’s known effects on Akt led us to investigate the response of this pathway in infarcted hearts exposed to SDF-1α. In harvested heart cell lysates from the infarct area, we observed increased levels of ILK protein and increased phosphorylation of its downstream kinase Akt upon SDF-1α treatment, while no difference was seen in the amount of total Akt protein. These changes were observed within 24 hours after coronary ligation (data not shown) and were more prominent at 72 hours (). Vascular endothelial growth factor (VEGF), a known regulator of angiogenesis, was similarly upregulated at 72 hours in response to SDF-1α, consistent with the increase in capillary density described in .
Figure 5 SDF-1α induces Akt activation and upregulation of VEGF. (A) Western blots with heart lysates 72 hours after coronary ligation and treatment with PBS or SDF-1α. ILK, upstream of Akt, was upregulated. Levels of Akt overall did not change (more ...)
To determine which cell types are direct targets of SDF-1α treatment, we treated primary cardiac-derived human microvascular endothelial cells (MVECs), primary human cardiac fibroblasts, a cardiac myocyte cell line (HL-1) and primary adult mouse cardiomyocytes in culture with SDF-1α. We found a modest but reproducible upregulation of phosphorylated Akt in MVECs and cardiomyocytes (HL-1 or primary adult cardiomyocytes), but not in fibroblasts (). These data suggest that SDF-1α may be acting on cardiomyocytes directly, but also on endothelial cells that may potentially signal to myocytes in a paracrine fashion. Consistent with this, we found that while CXCR4 protein was expressed in all of the cell types examined, it was most highly expressed in MVECs (data not shown). VEGF is one of the putative paracrine factors secreted from endothelial cells that functions in an angiogenic and cardioprotective manner. We observed an upregulation of VEGF protein in response to SDF-1α in MVECs, but not in cardiomyocytes. Thus, SDF-1α may function in a cardioprotective manner directly on cardiomyocytes and in a paracrine fashion through endothelial cells, in addition to stimulating new recruitment or expansion of the capillary bed.