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. 2010 Jan 5;5(1):e8576.
doi: 10.1371/journal.pone.0008576.

Sonic Hedgehog gene delivery to the rodent heart promotes angiogenesis via iNOS/netrin-1/PKC pathway

Rafeeq P H Ahmed  1 , Khawaja Husnain Haider, Jiang Shujia, Muhammad Rizwan Afzal, Muhammad Ashraf
Affiliations

Sonic Hedgehog gene delivery to the rodent heart promotes angiogenesis via iNOS/netrin-1/PKC pathway

Rafeeq P H Ahmed et al. PLoS One. .

Abstract

Background: We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway.

Methods/principal findings: MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid ((Shh)MSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in (Shh)MSCs which also led to increased expression of angiogenic and pro-survival growth factors in (Shh)MSCs. Significantly improved migration and tube formation was seen in (Shh)MSCs as compared to empty vector transfected MSCs ((Emp)MSCs). Significant upregulation of netrin-1 and iNOS was observed in (Shh)MSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 microl basal DMEM without cells (group-1) or containing 1x10(6) (Emp)MSCs (group-2) and (Shh)MSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of (Shh)MSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with (Shh)MSCs in group-3 animals.

Conclusions/significance: Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. In vitro characterization of ShhMSCs.
(A) RT-PCR and (B) Western blotting showed significant amplification of Shh transgene and Shh protein expression in ShhMSCs as compared with EmpMSCs on 72-h after transfection. (C) Fluorescent immunostaining of ShhMSCs for Shh overexpression (red fluorescence) at 72-h after trasnfection of Shh plasmid (magnification = 100x). (D) Western blot and (E) Fluorescent immunostaining revealed elevated expression of Ptc-1 in ShhMSCs (green fluorescence) as compared with EmpMSCs (magnification = 100x).
Figure 2
Figure 2. Expression of secretable angio-competent growth factors from ShhMSCs.
A. Shh overexpression in MSCs induced significant overexpression of multiple factors including Ang-1, VEGF, netrin-1 and iNOS as compared with EmpMSCs. (B) Invasion assay showing significantly higher migration of HUVECs in response to ShhCM as compared with EmpCM. (C) Culture of HUVECs in ShhCM promoted their differentiation into tubular network structures in vitro. Representative photomicrographs of HUVEC cultures for some experimental conditions as labeled in the individual photomicrograph (Magnification = 200x). (D) Quantitative analysis of network formation (formation of vascular assembly in culture) under the different experimental conditions expressed as network projections per low-power field. Results are the mean±SE from triplicate samples.
Figure 3
Figure 3. ShhMSCs upregulated iNOS and netrin-1 expression.
(A) RT-PCR and (B) Western blot showing significantly higher level iNOS gene and protein expression respectively in ShhMSCs as compared with EmpMSCs. (C) iNOS activity assay showed increased NO production (in 100-min/100 µg protein) in ShhMSCs as compared with EmpMSCs. This result was in accordance with higher level expression of iNOS in ShhMSCs thus indicating its functionally active status. (D) Transfection of ShhMSCs with Sc siRNA and PI3K specific siRNA failed to abrogate iNOS expression. On the other hand, pretreatment of ShhMSCs with 2.5 µM chel or 1 µM cyclopamine significantly blocked iNOS expression in ShhMSCs. (E) Western blot and densitometry of changes in PKM expression in ShhMSCs showed significantly higher level expression of 45 kDa fragment of PKC (PKM) in ShhMSCs as compared to EmpMSCs which was not blocked by PI3K specific RNA interference using Sc siRNA as a control. However, PKM fraction was sensitive to 2.5 µM chel or 1 µM cyclopamine. (F) Western blot showing significantly higher level protein expression of netrin-1 in ShhMSCs (Lanes-2 & 3) as compared with EmpMSCs (Lane-1). Netrin-1 expression was not abrogated by transfection of ShhMSCs with PI3K siRNA or Sc siRNA (Lanes-2 & 3). However, netrin-1 expression in ShhMSCs was abrogated by pretreatment of ShhMSCs with 2.5 µM chel (Lane-4) or 1 µM cyclopamine (Lane-5).
Figure 4
Figure 4. ShhMSCs survival in the infarcted heart.
(A) Real-time PCR for sry-gene showed improved survival of ShhMSCs in the infarcted rat heart on day-7 post engraftment as compared with EmpMSCs. Basal DMEM injected animal hearts showed no detection of sry-gene expression and served as a negative control. (B) FISH using rat y-chromosome specific probe labeled with red fluorescence to visualize transplanted male ShhMSCs on day-7 post-engraftment in group-3.
Figure 5
Figure 5. Shh overexpression attenuated infarction size expansion.
Masson's trichome staining of formalin fixed paraffin embedded of the histological sections from (A) group-1 (B) group-2 and (C) group-3 animal hearts was carried out to visualize the area of fibrosis. Infarction size was significantly attenuated in group-3 after ShhMSCs engraftment as compared with groups-1 and 2.
Figure 6
Figure 6. ShhMSCs improved blood vessel density in the infarcted heart.
(A–B) Blood vessel density analysis for myocardial angiogenesis at 8-weeks after respective treatment in different groups of animals. The histological sections were immunostained for vWFactor-VIII (red) and smooth muscle actin (green) for visualization of blood vessels. The number of blood vessels per surface area (0.74 mm2) was significantly higher in the infarct and peri-infarct areas in group-3 (p<0.05) as compared with group-1 and group-2. (C) The percentage of mature blood vessels (indicated by double fluorescent immunostaining for vWFactor-VIII and smooth muscle actin) showed no significant difference between the three treatment groups. However, (D–E) showed that average size of blood vessel diameter (based on number of pixels as arbitrary unit) was more uniform in peri-infarct region of group-3. Blood vessels in the infarct and peri-infarct areas with diameter of <100 pixels (33%), 100–200 pixels (39%) and >200 pixels (27%) diameter was observed in group-3 as compared with group-2 <100 (17%), 100–200 (43%) and >200 (39%). (F) Photomicrographs of hematoxylin-eosin stained histological sections at 8-weeks after their respective treatment in group-3 and group-2. Red blood cells could be seen in some of the blood vessels as indicated by green arrows showing the functional status of the blood vessels.
Figure 7
Figure 7. Recombinant netrin-1 treatment improved blood vessel density in the infarcted heart.
(A) At 8-weeks after recombinant netrin-1 delivery to the heart (group-4), the number of blood vessels per surface area (0.74 mm2) was significantly higher as compared with group-1 in both infarct as well as peri-infarct regions. (B) Double fluorescent immunostaining for vWFactor-VIII (red) and smooth muscle actin (green) showed that like Shh overexpression in the heart, netrin-1 protein delivery resulted in increased arteriolar density (blood vessels double positive for vWFactor-VIII and smooth muscle actin) in group-4. (C) Functional status of blood vessels in the infarcted heart was determined by fluorescent microsphere method for regional blood flow studies assessment. Regional blood flow was significantly improved in group-3 animal hearts as compared with group-1. However, regional blood flow changed insignificantly as compared with that of normal un-infarcted heart.
Figure 8
Figure 8. ShhMSCs transplantation preserved global function of the infarcted heart.
Echocardiographic assessment of the heart function indices (LVEF and LVFS) showed that preservation of the global heart function at 8 weeks after their respective treatment was significantly better in group-3 in comparison with DMEM injected group-1 and EmpMSCs transplanted group-2. LVEF and LVFS were calculated 1-week after myocardial infarction (before their respective treatment) and 8-weeks after respective treatment. Results are shown as mean±SEM (n = 7 animal per group).
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