Adipose-derived cells improve left ventricular diastolic function and increase microvascular perfusion in advanced age

Abstract

An early manifestation of coronary artery disease in advanced age is the development of microvascular dysfunction leading to deficits in diastolic function. Our lab has previously shown that epicardial treatment with adipose-derived stromal vascular fraction (SVF) preserves microvascular function following coronary ischemia in a young rodent model. Follow-up studies showed intravenous (i.v.) delivery of SVF allows the cells to migrate to the walls of small vessels and reset vasomotor tone. Therefore we tested the hypothesis that the i.v. cell injection of SVF would reverse the coronary microvascular dysfunction associated with aging in a rodent model. Fischer 344 rats were divided into 4 groups: young control (YC), old control (OC), old + rat aortic endothelial cells (O+EC) and old + GFP+ SVF cells (O+SVF). After four weeks, cardiac function and coronary flow reserve (CFR) were measured via echocardiography, and hearts were explanted either for histology or isolation of coronary arterioles for vessel reactivity studies. In a subgroup of animals, microspheres were injected during resting and dobutamine-stimulated conditions to measure coronary blood flow. GFP+ SVF cells engrafted and persisted in the myocardium and coronary vasculature four weeks following i.v. injection. Echocardiography showed age-related diastolic dysfunction without accompanying systolic dysfunction; diastolic function was improved in old rats after SVF treatment. Ultrasound and microsphere data both showed increased stimulated coronary blood flow in O+SVF rats compared to OC and O+EC, while isolated vessel reactivity was mostly unchanged. I.v.-injected SVF cells were capable of incorporating into the vasculature of the aging heart and are shown in this study to improve CFR and diastolic function in a model of advanced age. Importantly, SVF injection did not lead to arrhythmias or increased mortality in aged rats. SVF cells provide an autologous cell therapy option for treatment of microvascular and cardiac dysfunction in aged populations.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Diastolic function assessment using echocardiography.

The graphical representation of E/A ratio shows significant reduction of diastolic function in OC animals, which was rescued by SVF treatment;(A). The graphical representation of IVRT shows a significant increase of IVRT in OC and O+EC animals, which was rescued by SVF treatment; (B) P ≤ 0.05 vs. Old+SVF (*) and vs. Young Control (#); Data are presented as mean ± SD, analyzed with one-way ANOVA followed by Bonferroni post hoc test of following number of animals in each group: YC n = 9, OC n = 12, O+EC n = 10, O+SVF n = 8.

Fig 2. Measurement of coronary flow using Doppler echocardiography in rats.

Graphical representation of CFR calculation (A), Doppler velocity waveforms from the LAD were traced and peak velocity was measured during rest (scale bar = 850 mm/s) and during stress (Dobutamine infusion, scale bar = 1065 mm/s) (B). CFR was calculated from the peak velocities in our experimental groups (C). LAD peak velocity during rest (D) and Dobutamine-induced stress (E). P ≤ 0.05 vs. Old+SVF (*) and vs. Young Control (#); Data are presented as mean ± SD, analyzed with one-way ANOVA followed by Bonferroni post hoc test of following number of animals in each group: YC n = 10, OC n = 9, O+EC n = 10, O+SVF n = 9.

Fig 3. Measurement of heart perfusion using microspheres.

Coronary blood flow was evaluated by the injection of two different stable isotope-labeled microspheres during rest and dobutamine infusion. After explant, the LV was divided into 4 rings above the apex up to papillary region, then rings were separated by septum and free LV wall. Percent blood flow above resting for each section is depicted on the left, P ≤ 0.05 vs. O+SVF (*) and vs. OC (#) (A). Absolute LV blood flow (ml/min-1.g-1, averaged across all sections of one heart) during rest was significantly higher in OC compared to YC, p < .05 (#); following dobutamine challenge, O+SVF showed increased blood flow compared to OC and O+EC, p < .05 (*) (B). Data are presented as mean ± SD, analyzed with one-way ANOVA followed by Bonferroni post hoc test of the following number of animals in each group: YC n = 11, OC n = 11, O+EC n = 4, O+SVF n = 5.

Fig 4. Isolated coronary arteriole vasoreactivity.

Endothelin-induced vasoconstriction in coronary arterioles was similar between all groups (A). Coronary arterioles from O+EC or O+SVF decreased sensitivity to the low doses of bradykinin (O+EC group in 1e-13 and 1e-12 concentrations and O+SVF in 1e-12 and 1e-11 concentrations). Main group effect, P < 0.001, P ≤ 0.05 (YC and OC) vs. O+EC (*) vs O+SVF (#) (B). Myogenic response was higher in O+EC group during the 15 and 30mm Hg intramural pressure. Main group effect, P < 0.001, P ≤ 0.05 YC vs. O+EC (*) (C). Data are presented as mean ± SEM, analyzed with repeated measures ANOVA followed by Bonferroni post hoc test of the following number of animals in each group: Endothelin response: YC n = 9, OC n = 24, O+EC n = 9, O+SVF n = 15; Bradykinin response: YC n = 8, OC n = 18, O+EC n = 6, O+SVF n = 14; Myogenic response: YC n = 12, OC n = 23, O+EC n = 8, O+SVF n = 17.

Fig 5. Representative H&E staining of heart sections and trichrome analysis.

Examples of cross-sectional view of the whole hearts at the papillary level from the experimental groups, scale bar = 1mm (A). All old groups exhibited increased wall thickness compared to YC. SVF treatment significantly decreased wall thickness compared to OC (B). Main group effect P P < 0.05 vs. Young Control (*) and vs. Old Control (#). H&E data are analyzed with one-way ANOVA followed by Bonferroni post hoc test of following number of animals in each group: YC n = 4, OC n = 3, O+EC n = 3, O+SVF n = 7. Masson’s trichrome staining revealed an increase in % collagen content in OC compared to YC (C). Main group effect P = 0.010, P < 0.05 vs. Young Control (*). Trichrome data are analyzed with Kruskall-Wallis one-way ANOVA, YC: n = 3, OC n = 3, O+SVF n = 4. Data are presented as mean ± SD.

Fig 6. GFP positive cells in different organs four weeks following GFP+ SVF cell injection.

GFP+ cells were found in aorta, carotid artery, lung, brain and heart, scale bar = 100μm (A). Graphical representation of percentage of GFP+ cells to total number of nucleated cells (DAPI, blue) in different organs (B). Co-localization of GFP (green), ED1 (macrophage marker, red), and NG2 (pericyte marker, orange) in heart, scale bar = 50μm and 20μm in the insert image (C). YC n = 4, OC n = 3, O+EC n = 3, O+SVF n = 7.