Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis

A new era has begun in the treatment of ischemic disease and heart failure. With the discovery that stem cells from diverse organs and tissues, including bone marrow, adipose tissue, umbilical cord blood, and vessel wall, have the potential to improve cardiac function beyond that of conventional pharmacological therapy comes a new field of research aiming at understanding the precise mechanisms of stem cell-mediated cardiac repair. Not only will it be important to determine the most efficacious cell population for cardiac repair, but also whether overlapping, common mechanisms exist. Increasing evidence suggests that one mechanism of action by which cells provide tissue protection and repair may involve paracrine factors, including cytokines and growth factors, released from transplanted stem cells into the surrounding tissue. These paracrine factors have the potential to directly modify the healing process in the heart, including neovascularization, cardiac myocyte apoptosis, inflammation, fibrosis, contractility, bioenergetics, and endogenous repair

Interleukin-10 From Transplanted Bone Marrow Mononuclear Cells Contributes to Cardiac Protection After Myocardial Infarction

Bone marrow mononuclear cells (BM-MNCs) have successfully been used as a therapy for the improvement of left ventricular (LV) function after myocardial infarction (MI). It has been suggested that paracrine factors from BM-MNCs may be a key mechanism mediating cardiac protection. We previously performed microarray analysis and found that the pleiotropic cytokine interleukin (IL)-10 was highly upregulated in human progenitor cells in comparison with adult endothelial cells and CD14+cells. Moreover, BM-MNCs secrete significant amounts of IL-10, and IL-10 could be detected from progenitor cells transplanted in infarcted mouse hearts. Specifically, intramyocardial injection of wild-type BM-MNCs led to a significant decrease in LV end-diastolic pressure (LVEDP) and LV end-systolic volume (LVESV) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. Furthermore, intramyocardial injection of wild-type BM-MNCs led to a significant increase in stroke volume (SV) and rate of the development of pressure over time (+dP/dt) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. The IL-10–dependent improvement provided by transplanted cells was not caused by reduced infarct size, neutrophil infiltration, or capillary density, but rather was associated with decreased T lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. These results suggest that BM-MNCs mediate cardiac protection after myocardial infarction and this is, at least in part, dependent on IL-10

Optimizing Recellularization of Whole Decellularized Heart Extracellular Matrix

<div><p>Rationale</p><p>Perfusion decellularization of cadaveric hearts removes cells and generates a cell-free extracellular matrix scaffold containing acellular vascular conduits, which are theoretically sufficient to perfuse and support tissue-engineered heart constructs. However, after transplantation, these acellular vascular conduits clot, even with anti-coagulation. Here, our objective was to create a less thrombogenic scaffold and improve recellularized-left ventricular contractility by re-lining vascular conduits of a decellularized rat heart with rat aortic endothelial cells (RAECs).</p><p>Methods and Results</p><p>We used three strategies to recellularize perfusion-decellularized rat heart vasculature with RAECs: retrograde aortic infusion, brachiocephalic artery (BA) infusion, or a combination of inferior vena cava (IVC) plus BA infusion. The re-endothelialized scaffolds were maintained under vascular flow <i>in vitro</i> for 7 days, and then cell morphology, location, and viability were examined. Thrombogenicity of the scaffold was assessed <i>in vitro</i> and <i>in vivo</i>. Both BA and IVC+BA cell delivery resulted in a whole heart distribution of RAECs that proliferated, retained an endothelial phenotype, and expressed endothelial nitric oxide synthase and von Willebrand factor. Infusing RAECs via the combination IVC+BA method increased scaffold cellularity and the number of vessels that were lined with endothelial cells; re-endothelialization by using BA or IVC+BA cell delivery significantly reduced <i>in vitro</i> thrombogenicity. <i>In vivo</i>, both acellular and re-endothelialized scaffolds recruited non-immune host cells into the organ parenchyma and vasculature. Finally, re-endothelialization before recellularization of the left ventricular wall with neonatal cardiac cells enhanced construct contractility.</p><p>Conclusions</p><p>This is the first study to re-endothelialize whole decellularized hearts throughout both arterial and venous beds and cavities by using arterial and venous delivery. The combination (IVC+BA) delivery strategy results in enhanced scaffold vessel re-endothelialization compared to single-route strategies. Re-endothelialization reduced scaffold thrombogencity and improved contractility of left ventricular-recellularized constructs. Thus, vessel and cavity re-endothelialization creates superior vascularized scaffolds for use in whole-organ recellularization applications.</p></div

Histologic assessment of decellularized rat heart scaffolds seeded with RAECs.

<p>(A,C) H&E and (B,D) Verhoeff-Van Gieson staining of scaffolds recellularized by the BA only technique (A,B) and the combined IVC+BA technique (C,D); arrows indicate cell-free vessels (A), arrow heads indicate elastin-positive vessels with cell nuclei (B,D), and open arrow heads indicate elastin-negative vessels (B,D). All scaffolds were recellularized with 4×10⁷ RAECs. (E) Quantification of the number of vessels lined by DAPI-positive cell nuclei in the mid-ventricular wall; the results are grouped according to vessel diameter (n = 3 per data set; mean ±SEM). **p<0.001 for IVC+BA vs BA re-endothelialization techniques for vessels with a diameter of 11–25 microns. Scale bars represent 125 microns (A–D).</p

Cellularity and localization of labeled RAECs.

<p>(A) Number of DAPI-positive nuclei per mm² of scaffold for each delivery method: the aorta only (Aorta), the BA only (BA), or the combined IVC+BA method (n = 3 hearts per method; mean ±SEM). The total number of cells delivered is indicated in parenthesis. (B, C) Image of whole heart in which 4×10⁷ DiI-labeled RAECs were delivered via the BA. (D, E) Image of whole heart in which 2×10⁷ DiO-labeled cells were delivered via the IVC followed by an additional 2×10⁷ DiI-labeled cells administered via the BA. View of the (F) left ventricular endocardial surface, (G) right ventricular endocardial surface, and (H) the ventricle wall of a heart scaffold recellularized via the BA+IVC cell delivery technique with cells labeled as in D and E. DAPI-positive nuclei are blue (F–H), and overlapping green and red staining shows as yellow (D–H). *p<0.05. Scale bars represent 5 mm (B–E) and 50 microns (F).</p

Cell survival in re-endothelialized heart scaffolds.

<p>(A) CMFDA-labeled cells (green) in the ventricle wall of a scaffold recellularized with 4×10⁷ RAECs via the BA technique and cultured for seven days before CMFDA labeling.(B) Quantification of G6PDH activity in the medium as an indicator of cell viability over time expressed as a percent of the initial relative fluorescence unit (RFU) measured using the Vybrant Cytotoxicity Assay Kit (n = 6 for each re-endothelialization technique; results are expressed as mean ±SEM).(C–H) TUNEL staining of scaffolds re-endothelialized with 4×10⁷ RAECs after seven days of culture. Images of the (C) left ventricle (LV), (D) septum, and (E) right ventricle (RV) of scaffolds seeded using the BA cell delivery technique. Images of the (F) left ventricle, (G) septum, and (H) right ventricle of scaffolds seeded using the IVC+BA cell delivery technique. (C–H) Cell nuclei are stained with DAPI (blue), and TUNEL-positive staining is red (arrows). Scale bars represent 100 microns.</p

Characterization of heterotopic transplants.

<p>(A–E) Acellular and (F–J) re-endothelialized scaffolds seven days after heterotopic transplantation. Short axial view of an aorta with blood clot (arrow) from an acellular scaffold (A) and a non-clotted aorta (arrow) from a re-endothelialized scaffold (F) after transplantation. Short axial view of the ventricle wall of an acellular scaffold with a blood clot (B) and a re-endothelialized scaffold (G). H&E staining of a transplanted acellular scaffold (C–E) with a blood clot inside the ventricular cavity and a re-endothelialized scaffold (H–J) at increasing magnification (2X, 10X, and 20X). Arrows point to patent vessels with and without blood (J). CD31 (K) and VEGFR2 (L) (red) staining in transplanted scaffolds; DAPI-positive nuclei are blue. Scale bars represent 1 mm (A–C and F–H) and 100 microns (D, E, I, J, K, and L).</p

Functional analysis of re-endothelialized scaffolds after seven days of cell culture.

<p>Scaffolds recellularized via the BA showing (A) CMFDA (green) and PCNA (red) staining; (B) CMFDA (green), eNOS (red), and DAPI (blue) staining; and (C) CMFDA (green), vWF (red), and DAPI (blue) staining; yellow staining indicates the combination of green and red. (D) Thrombogenicity of re-endothelialized matrices expressed as a ratio of recellularized to acellular controls (n = 6 for acellular controls, n = 8 for BA and BA+IVC re-endothelialized scaffolds). The total number of RAECs delivered is indicated in parentheses. *p<0.05, re-endothelialized scaffolds vs acellular controls; results are expressed as the mean ±SEM. Scale bars represent 100 microns (A–C).</p

Effect re-endothelialization before left ventricle wall recellularization on contractility of the heart construct.

<p>(A) Maximal rate of change in pressure (dP/dt max) of the left ventricle at different pacing frequencies in constructs that were re-endothelialized with rat aortic endothelial cells and recellularized with rat neonatal cardiac cells (RAEC+CM) and control constructs in which neonatal cardiac cells were injected into the left ventricle wall without prior re-endothelialization (CM only). (B–C) H&E staining of rat neonatal cardiac cells in the left ventricle of a scaffold without (B) and with re-endothelialization (C). Arrows indicate re-lined vessels in the recellularized left ventricle. Scale bars represent 100 micron; n = 3 in each group; *p<0.05, vs control constructs; the results are expressed as the mean ±SEM.</p