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. 2013 May;93(5):577-91.
doi: 10.1038/labinvest.2013.48. Epub 2013 Mar 18.

Tissue kallikrein-modified human endothelial progenitor cell implantation improves cardiac function via enhanced activation of akt and increased angiogenesis

Yuyu Yao  1 , Zulong Sheng, YeFei Li, Cong Fu, Genshan Ma, Naifeng Liu, Julie Chao, Lee Chao
Affiliations

Tissue kallikrein-modified human endothelial progenitor cell implantation improves cardiac function via enhanced activation of akt and increased angiogenesis

Yuyu Yao et al. Lab Invest. 2013 May.

Abstract

Endothelial progenitor cells (EPCs) have been shown to enhance angiogenesis not only by incorporating into the vasculature but also by secreting cytokines, thereby serving as an ideal vehicle for gene transfer. As tissue kallikrein (TK) has pleiotropic effects in inhibiting apoptosis and oxidative stress, and promoting angiogenesis, we evaluated the salutary potential of kallikrein-modified human EPCs (hEPCs; Ad.hTK-hEPCs) after acute myocardial infarction (MI). We genetically modified hEPCs with a TK gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a nude mouse left anterior descending ligation model. hEPCs were manipulated to overexpress the TK gene. In vitro, the antiapoptotic and paracrine effects were assessed under oxidative stress. TK protects hEPCs from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and -9, induction of Akt phosphorylation, and secretion of vascular endothelial growth factor. In vivo, the Ad.hTK-hEPCs were transplanted after MI via intracardiac injection. The surviving cells were tracked after transplantation using near-infrared optical imaging. Left ventricular (LV) function was evaluated by transthoracic echocardiography. Capillary density was quantified using immunohistochemical staining. Engrafted Ad.hTK-hEPCs exhibited advanced protection against ischemia by increasing LV ejection fraction. Compared with Ad.Null-hEPCs, transplantation with Ad.hTK-hEPCs significantly decreased cardiomyocyte apoptosis in association with increased retention of transplanted EPCs in the myocardium. Capillary density and arteriolar density in the infarct border zone was significantly higher in Ad.hTK-hEPC-transplanted mice than in Ad.Null-hEPC-treated mice. Transplanted hEPCs were clearly incorporated into CD31(+) capillaries. These results indicate that implantation of kallikrein-modified EPCs in the heart provides advanced benefits in protection against ischemia-induced MI by enhanced angiogenesis and reducing apoptosis.

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Conflict of interest statement

Conflict of Interest. The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of cultured hEPCs. (A) At day 7 after hEPCs isolation, adherent cells intensively took up acLDL-Dil and bound endothelial-specific lectin-FITC as revealed by fluorescence microscopy. (B) Characterization of hEPCs evaluated by flow cytometry. EPCs were positive for CD34 (55.4%), CDKDR (49.3%) and kinin B2 receptor (43.2%). (C). Expression of human tissue kallikrein (hTK) in hEPCs after Ad.hTK transduction was confirmed by immunocytochemistry. Original magnification is 200 ×.
Figure 2
Figure 2
TK reduced EPC apoptosis induced by H2O2. (A) Representative flow cytometric analysis of apoptosis showed that TK gene transfer protects EPCs from H2O2-induced apoptosis. (B). In situ TUNEL assay was applied to examine cultured hEPC apoptosis (upper panel), lower panels showed staining of corresponding sections with the nuclear stain DAPI. (C) Quantitative analysis of EPC apoptosis by flow cytometry (*P<0.05 vs. other groups, n=4). (D). Quantitative analysis of EPC apoptosis by TUNEL staining (*P<0.05 vs. other groups, n=3). (E) Western blots for Akt and cleaved caspased-3. (F, G) Activation of caspase-3, caspase-9 levels were significantly increased by exposure to H2O2, TK gene transfer inhibited caspase-3, 9 activities. Results are the mean ± SEM from three independent experiments (*P <0.01, respectively; n=3). (H). VEGF secretion by Ad.TK-hEPCs under oxidative stress. After 24 h of incubation, conditioned medium from control and treated cells (n= 3) was subjected to VEGF ELISA assay. VEGF concentration values are mean ± SEM (*P<0.05). ELISA data are representative of three independent experiments. (I). Effect of the EPCs conditioned medium on apoptosis of neonatal rat ventricular cardiomyocytes exposed to oxidative stress. Representative Hoechst 33324 staining images in neonatal rat ventricular cardiomyocytes treated with 0.2 mM H2O2 for 12 h. Hoechst 33324 staining for nuclear morphology was performed to assess apoptotic cell death. Lower panel showed quantitative analysis of apoptosis levels using Hoechst 33324 staining in control medium, hypoxic Ad.Null-hEPCs conditioned medium and hypoxic Ad.TK-hEPCs conditioned medium 12 h after H2O2. (*P<0.05 vs. control medium, hypoxic Ad.Null-hEPCs conditioned medium; #P<0.05 vs. control medium, n=4). Original magnification is 200 ×.
Figure 2
Figure 2
TK reduced EPC apoptosis induced by H2O2. (A) Representative flow cytometric analysis of apoptosis showed that TK gene transfer protects EPCs from H2O2-induced apoptosis. (B). In situ TUNEL assay was applied to examine cultured hEPC apoptosis (upper panel), lower panels showed staining of corresponding sections with the nuclear stain DAPI. (C) Quantitative analysis of EPC apoptosis by flow cytometry (*P<0.05 vs. other groups, n=4). (D). Quantitative analysis of EPC apoptosis by TUNEL staining (*P<0.05 vs. other groups, n=3). (E) Western blots for Akt and cleaved caspased-3. (F, G) Activation of caspase-3, caspase-9 levels were significantly increased by exposure to H2O2, TK gene transfer inhibited caspase-3, 9 activities. Results are the mean ± SEM from three independent experiments (*P <0.01, respectively; n=3). (H). VEGF secretion by Ad.TK-hEPCs under oxidative stress. After 24 h of incubation, conditioned medium from control and treated cells (n= 3) was subjected to VEGF ELISA assay. VEGF concentration values are mean ± SEM (*P<0.05). ELISA data are representative of three independent experiments. (I). Effect of the EPCs conditioned medium on apoptosis of neonatal rat ventricular cardiomyocytes exposed to oxidative stress. Representative Hoechst 33324 staining images in neonatal rat ventricular cardiomyocytes treated with 0.2 mM H2O2 for 12 h. Hoechst 33324 staining for nuclear morphology was performed to assess apoptotic cell death. Lower panel showed quantitative analysis of apoptosis levels using Hoechst 33324 staining in control medium, hypoxic Ad.Null-hEPCs conditioned medium and hypoxic Ad.TK-hEPCs conditioned medium 12 h after H2O2. (*P<0.05 vs. control medium, hypoxic Ad.Null-hEPCs conditioned medium; #P<0.05 vs. control medium, n=4). Original magnification is 200 ×.
Figure 2
Figure 2
TK reduced EPC apoptosis induced by H2O2. (A) Representative flow cytometric analysis of apoptosis showed that TK gene transfer protects EPCs from H2O2-induced apoptosis. (B). In situ TUNEL assay was applied to examine cultured hEPC apoptosis (upper panel), lower panels showed staining of corresponding sections with the nuclear stain DAPI. (C) Quantitative analysis of EPC apoptosis by flow cytometry (*P<0.05 vs. other groups, n=4). (D). Quantitative analysis of EPC apoptosis by TUNEL staining (*P<0.05 vs. other groups, n=3). (E) Western blots for Akt and cleaved caspased-3. (F, G) Activation of caspase-3, caspase-9 levels were significantly increased by exposure to H2O2, TK gene transfer inhibited caspase-3, 9 activities. Results are the mean ± SEM from three independent experiments (*P <0.01, respectively; n=3). (H). VEGF secretion by Ad.TK-hEPCs under oxidative stress. After 24 h of incubation, conditioned medium from control and treated cells (n= 3) was subjected to VEGF ELISA assay. VEGF concentration values are mean ± SEM (*P<0.05). ELISA data are representative of three independent experiments. (I). Effect of the EPCs conditioned medium on apoptosis of neonatal rat ventricular cardiomyocytes exposed to oxidative stress. Representative Hoechst 33324 staining images in neonatal rat ventricular cardiomyocytes treated with 0.2 mM H2O2 for 12 h. Hoechst 33324 staining for nuclear morphology was performed to assess apoptotic cell death. Lower panel showed quantitative analysis of apoptosis levels using Hoechst 33324 staining in control medium, hypoxic Ad.Null-hEPCs conditioned medium and hypoxic Ad.TK-hEPCs conditioned medium 12 h after H2O2. (*P<0.05 vs. control medium, hypoxic Ad.Null-hEPCs conditioned medium; #P<0.05 vs. control medium, n=4). Original magnification is 200 ×.
Figure 2
Figure 2
TK reduced EPC apoptosis induced by H2O2. (A) Representative flow cytometric analysis of apoptosis showed that TK gene transfer protects EPCs from H2O2-induced apoptosis. (B). In situ TUNEL assay was applied to examine cultured hEPC apoptosis (upper panel), lower panels showed staining of corresponding sections with the nuclear stain DAPI. (C) Quantitative analysis of EPC apoptosis by flow cytometry (*P<0.05 vs. other groups, n=4). (D). Quantitative analysis of EPC apoptosis by TUNEL staining (*P<0.05 vs. other groups, n=3). (E) Western blots for Akt and cleaved caspased-3. (F, G) Activation of caspase-3, caspase-9 levels were significantly increased by exposure to H2O2, TK gene transfer inhibited caspase-3, 9 activities. Results are the mean ± SEM from three independent experiments (*P <0.01, respectively; n=3). (H). VEGF secretion by Ad.TK-hEPCs under oxidative stress. After 24 h of incubation, conditioned medium from control and treated cells (n= 3) was subjected to VEGF ELISA assay. VEGF concentration values are mean ± SEM (*P<0.05). ELISA data are representative of three independent experiments. (I). Effect of the EPCs conditioned medium on apoptosis of neonatal rat ventricular cardiomyocytes exposed to oxidative stress. Representative Hoechst 33324 staining images in neonatal rat ventricular cardiomyocytes treated with 0.2 mM H2O2 for 12 h. Hoechst 33324 staining for nuclear morphology was performed to assess apoptotic cell death. Lower panel showed quantitative analysis of apoptosis levels using Hoechst 33324 staining in control medium, hypoxic Ad.Null-hEPCs conditioned medium and hypoxic Ad.TK-hEPCs conditioned medium 12 h after H2O2. (*P<0.05 vs. control medium, hypoxic Ad.Null-hEPCs conditioned medium; #P<0.05 vs. control medium, n=4). Original magnification is 200 ×.
Figure 3
Figure 3
TK-modified hEPC transplantation reduced infarct size. (A) Expression of human TK in hEPCs after Ad.hTK transduction was confirmed by immunocytochemistry (left); the same view of DiD-labeled cells all appeared in red under fluorescence microscopy (× 100; Excitation wavelength coverage 595-800nm, Emission coverage 660-680nm). (B) Fluorescent images show increasing signal with increasing cell number in hEPCs. (C) Correlation plot shows robust correlation between signal and cell number (R2=0.9977). (D) Representative immunofluorescence photographs of DiD-labeled Ad.TK-hEPC transplantation group (red) and TK protein expression (green) in the infarcted region of mouse hearts 2 days after MI. Low panel is DiD-labeled Ad.Null-hEPC transplantation group (red). Original magnification is 600 ×. (E) Representative Masson’s trichrome staining. Original magnification is 10 ×. (F) Echocardiographic measurements for determination of left ventricular function from M-mode measurements. (G) MDA in the ischemic mouse heart at day 7 after MI, Values are expressed as mean ± SEM (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated, medium-treated, #P < 0.05 vs. Medium-treated)
Figure 3
Figure 3
TK-modified hEPC transplantation reduced infarct size. (A) Expression of human TK in hEPCs after Ad.hTK transduction was confirmed by immunocytochemistry (left); the same view of DiD-labeled cells all appeared in red under fluorescence microscopy (× 100; Excitation wavelength coverage 595-800nm, Emission coverage 660-680nm). (B) Fluorescent images show increasing signal with increasing cell number in hEPCs. (C) Correlation plot shows robust correlation between signal and cell number (R2=0.9977). (D) Representative immunofluorescence photographs of DiD-labeled Ad.TK-hEPC transplantation group (red) and TK protein expression (green) in the infarcted region of mouse hearts 2 days after MI. Low panel is DiD-labeled Ad.Null-hEPC transplantation group (red). Original magnification is 600 ×. (E) Representative Masson’s trichrome staining. Original magnification is 10 ×. (F) Echocardiographic measurements for determination of left ventricular function from M-mode measurements. (G) MDA in the ischemic mouse heart at day 7 after MI, Values are expressed as mean ± SEM (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated, medium-treated, #P < 0.05 vs. Medium-treated)
Figure 4
Figure 4
Ex-vivo optical imaging study. (A, B) Representative NIR fluorescent images in explanted organs 2 or 7 days following implantation of DiD-labeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (A: Two days after cell delivery. B: Seven days after cell delivery). (C) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean ± SEM (n=3-4, *P <0.01 vs. control group).
Figure 5
Figure 5
Confocal immunofluorescent images of DiD-labeled hEPCs and TUNEL- positive cardiomyocytes. (A) TUNEL-positive cardiomyocytes (green) and NIR fluorescent signals (red) were co-observed in the myocardium at day 2; counterstaining was performed with DAPI (blue). Original magnification is 600 ×. (B) Representative micrographs show DiD-positive cells in mice heart at 7 days after MI (red, 400 × magnification). Nuclei were counterstained with DAPI (blue). (C) Quantitative analysis of apoptotic cardiomyocytes expressed as percentage of TUNEL-positive nuclei in cardiomyocytes. TUNEL-positive non-cardiomyocytes were excluded (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated, medium-treated; #P<0.05 vs. medium-treated). (D) Quantitative analysis of DiD-positive cells in mice heart at 2 days after MI (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated).
Figure 5
Figure 5
Confocal immunofluorescent images of DiD-labeled hEPCs and TUNEL- positive cardiomyocytes. (A) TUNEL-positive cardiomyocytes (green) and NIR fluorescent signals (red) were co-observed in the myocardium at day 2; counterstaining was performed with DAPI (blue). Original magnification is 600 ×. (B) Representative micrographs show DiD-positive cells in mice heart at 7 days after MI (red, 400 × magnification). Nuclei were counterstained with DAPI (blue). (C) Quantitative analysis of apoptotic cardiomyocytes expressed as percentage of TUNEL-positive nuclei in cardiomyocytes. TUNEL-positive non-cardiomyocytes were excluded (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated, medium-treated; #P<0.05 vs. medium-treated). (D) Quantitative analysis of DiD-positive cells in mice heart at 2 days after MI (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated).
Figure 6
Figure 6
TK-modified hEPCs transplantation increased capillary density and arteriole density 7 days after MI. Representative photographs of immunostaining using (A) CD31 to identify capillaries and (B) α-SMA to identify arterioles. Original magnification is 200 ×. Quantitative analysis of (C) capillary density and (D) arteriole density in the peri-infarct myocardium (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated, medium-treated; #P<0.05 vs. medium-treated). (E) High power field of DiD-labeled implanted hEPCs (red) and immunofluorescent staining of CD31 or α-SMA (green) at the border zone of the ischemic myocardium; nuclei were counterstained with DAPI (blue). Original magnification is 1200 ×. Transplanted DiD–labeled hEPCs were clearly incorporated into CD31+ capillaries and α-SMC+ small arteries in the Ad.TK-hEPCs group.
Figure 6
Figure 6
TK-modified hEPCs transplantation increased capillary density and arteriole density 7 days after MI. Representative photographs of immunostaining using (A) CD31 to identify capillaries and (B) α-SMA to identify arterioles. Original magnification is 200 ×. Quantitative analysis of (C) capillary density and (D) arteriole density in the peri-infarct myocardium (n=6, *P<0.05 vs. Ad.Null-hEPCs-treated, medium-treated; #P<0.05 vs. medium-treated). (E) High power field of DiD-labeled implanted hEPCs (red) and immunofluorescent staining of CD31 or α-SMA (green) at the border zone of the ischemic myocardium; nuclei were counterstained with DAPI (blue). Original magnification is 1200 ×. Transplanted DiD–labeled hEPCs were clearly incorporated into CD31+ capillaries and α-SMC+ small arteries in the Ad.TK-hEPCs group.
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