Angiogenesis and myogenesis using human skeletal myoblasts for cardiac repair 2

Dual fluorescent immunostaining demonstrated that >95% of skeletal myoblasts simultaneously expressed desmin and CD56 Figure 2c using human fibroblasts as a negative control Figure 2d..

Chapter 3 Results

3.1 In vitro characterization of skeletal myoblast

Fresh culture of human skeletal myoblasts was obtained from Cell Transplants Singapore Pte Ltd Singapore, for every transplantation experiment The cells were grown and cultured in the laboratory using Super-medium to achieve the required number

3.1.1 Cell culture characteristics of skeletal myoblast

Skeletal myoblasts obtained from human male donor had a typical morphological appearance Freshly cultured human skeletal myoblasts were small spheres of about 12 µm

in diameter (Figure 1a) They transformed into elongated, spindle-shaped cells and started

to grow as an adherent culture after 12 hours on collagen coated tissue culture flasks (Figure 1b) They reached to 50% of confluence at 48 hours (Figure 1c) and > 90% of confluence at 120 hours (Figure 1d) after isolation Their doubling time cultured in Super-medium supplemented with 10% FBS, incubated at 37oC in 5% CO2 incubator was less than 18 hours Skeletal myoblasts were regularly passaged every 48-72 hours to prevent their pre-mature fusion and differentiation in vitro

3.1.2 Purity of skeletal myoblast culture

Skeletal myoblast culture was more than 98 % of purity as shown by desmin or CD56 expression (Figure 2a & b) Dual fluorescent immunostaining demonstrated that

>95% of skeletal myoblasts simultaneously expressed desmin and CD56 (Figure 2c) using human fibroblasts as a negative control (Figure 2d)

Cytofluorimetric study further confirmed high purity of skeletal myoblast culture The result showed that 96% of skeletal myoblasts were positive for desmin expression when 1% of autofluorescence was gated by non-stained skeletal myoblasts (Figure 3a &

c

d

Figure 1a-d: Phase contrast photomicrographs of human skeletal myoblasts showing in

d

c

Figure 2a-d: Dual fluorescent immunostaining of human skeletal myoblasts for desmin

and CD56 expression (a) Desmin expression (green fluorescence = FITC) (b) CD56 expression (red fluorescence = PE) (c) Superimposition of pictures (a) and (b) to show co- expression of desmin and CD56 (d) Human fibroblasts were used as a negative control

The cells were counterstained with DAPI (blue fluorescence) to show the nuclei (Magnification = 200x)

Figure 3a-i: Cytofluorimetry of human skeletal myoblasts for desmin and CD56

expression Non-stained human skeletal myoblasts (missing primary antibody) for (a)

desmin (d) CD56 and (g) co-expression of desmin and CD56 were used as a baseline for

autofluorescence The results showed that (b) 96% of skeletal myoblasts expressed desmin

whereas (e) 92% of skeletal myoblast expressed CD56, while (h) 91% of skeletal

myoblasts co-expressed desmin and CD56 Human fibroblasts were used as negative

controls (c) for desmin (f) for CD56 and (i) for co-expression of desmin and CD56

3.1.3 The effect of multiple transduction on skeletal myoblast

Fluorescent immunostaining and cytofluorimetric analysis for desmin and CD56 expression demonstrated that viral vector transduction with skeletal myoblasts did not change the phenotype of skeletal myoblasts Dual fluorescent immunostaining showed that

>95% of skeletal myoblasts expressed desmin or CD56, or co-expressed desmin and CD56 (Figure 4a-c) Cytofluorimetric analysis further confirmed these results It was observed that 83% of skeletal myoblasts expressed desmin (Figure 5b), while 95% of them expressed CD56 (Figure 5d) when 1% of autofluorescence was gated by non-stained skeletal myoblasts (Figure 5a & c) Co-expression of desmin and CD56 was observed to be 82% (Figure 5f)

3.1.4 Skeletal myoblast viability

Cell viability at the time of cell transplantation was more than 99% as determined

by Trypan blue dye exclusion test

3.2 Ad-vector titer

The adenoviral vector titer determined by end-point assay revealed that the viral titer was ~1x1010 PFU/ ml for Ad-null, ~8x109 PFU/ ml for Ad-VEGF165, ~7x109 PFU/ ml for Ad-Ang-1 and ~5x108 PFU/ ml for Ad-Bic All of the viral vectors were found replication deficient when tested for replication competence

3.3 Skeletal myoblast transduction with Ad-VEGF 165

After optimizing the transduction procedure, qualitative and quantitative

d

c

Figure 4a-d: Dual fluorescent immunostaining of human skeletal myoblasts for desmin

and CD56 expression after multiple viral vector transduction (a) Desmin expression (green fluorescence = FITC), (b) CD56 expression (red fluorescence = PE) (c) Superimposition of pictures (a) and (b) to show co-expression of desmin and CD56 (d)

Human fibroblasts were used as a negative control The cells were counterstained with DAPI (blue fluorescence) to show the nuclei (Magnification = 200x)

Figure 5 a-f: Cytofluorimetry of human skeletal myoblasts for desmin and CD56

expression Non-stained human skeletal myoblasts (missing primary antibody) for (a)

3.3.1 Optimization of transduction condition

A dose dependent relation between the Ad-VEGF165 vector titer and skeletal myoblast number was revealed by human VEGF ELISA With the increase in the number

of viral particles, transduction efficiency also increased (Figure 6a) The highest transduction efficiency was, however, achieved at 1000PFU/ myoblast The exposure time

of skeletal myoblast to Ad-VEGF165 vector showed a causal relation with transduction efficiency (Figure 6b) Repeated transduction (in triplicate) of skeletal myoblasts improved transduction efficiency (Figure 6b) At higher viral titer (>1000 PFU) or a longer time (>8 hours) exposure, however, the ill effects of adenoviral vector towards skeletal myoblasts were observed Thus, the optimum transduction conditions were achieved at a ratio of 1000PFU/ myoblast when transduction was carried out for 8 hours in triplicate with 24 hours interval

3.3.2 Qualitative assessment of hVEGF 165 expression from hVEGF 165 -myoblasts

Immunohistochemical staining of hVEGF165-myoblasts revealed high transduction efficiency Both the DAB substrate (Figure 7a & c) and FITC fluorescent (Figure 7d) immunostaining showed > 95% of Ad-VEGF165 transduced myoblasts expressing hVEGF165, using Ad-null transduced myoblasts as a negative control (Figure 7b & e) The strong signal demonstrated that hVEGF165 was actively expressed by Ad-VEGF165transduced myoblasts RT-PCR for hVEGF165 expression revealed that Ad-VEGF165transduced myoblasts expressed hVEGF165 for up to 30 days of observation in vitro (Figure 8) The presence of mRNA encoding for hVEGF165 as detected by hVEGF165 specific primers demonstrated that Ad-VEGF165 transduced myoblasts had the highest level of hVEGF gene expression compared with non-transduced and Ad-null transduced

0 500 1000 1500 2000

PBS transduced myoblast

Non-Ad-null myoblast

Figure 6a & b: Optimization of skeletal myoblast transduction conditions using

Ad-VEGF165 (a) Efficiency of skeletal myoblast transduction as a function of Ad-VEGF165titer Optimum transduction efficiency was achieved at a ratio of 1000 Ad-vector : one

myoblast (b) Expression of hVEGF165 from hVEGF165-myoblast as a function of transduction time Transduction was repeated three times

Figure 7a-d: Immunostaining of Ad-VEGF165 transduced myoblasts for hVEGF165

expression (a) hVEGF165 was visualized as brown color using DAB substrate detection

system using (b) Ad-null transduced myoblasts were used as a negative control (c) A

typical human skeletal myoblasts expressing hVEGF165 in vitro (d) Fluorescent

immunostaining for hVEGF165 expression as green fluorescence (FITC) using (e) Ad-null

1 day 8 days 18 days 30 days

Figure 8: RT-PCR of Ad-VEGF165 transduced myoblasts for hVEGF165 expression

Lane 1 = DNA ladder

Lane 2 & 3 = Non-transduced myoblasts

Lane 4 & 5 = Ad-null transduced myoblasts

Lane 6 ~13 = Ad-VEGF165 transduced myoblasts at 1, 8, 18 and 30 days

after transduction

Western blot analysis of supernatant and cell lysate, from Ad-VEGF165 transduced myoblasts, showed the presence of hVEGF165 in the supernatant as well as in the cell lysate (Figure 9) The presence of hVEGF165 in the supernatant and cell lysate demonstrated that hVEGF165 was expressed by transduced myoblasts The molecular weight of secreted hVEGF165 from hVEGF165-myoblasts was 42 kDa

3.3.3 Quantitative assessment of hVEGF 165 expression from hVEGF 165 -myoblasts

ELISA results showed that skeletal myoblasts transduced under optimum transduction condition secreted hVEGF165 in vitro at least for up to 30 days of observation (Figure 10) The peak level of secreted hVEGF165 (37 ± 3 ng/ ml) in cell-cultured supernatant was achieved at day 8 after transduction However, the non-transduced myoblasts and Ad-null myoblasts secreted hVEGF165 at very low level (0.3 ± 0.05 ng/ ml and 0.8 ± 0.1 ng/ ml respectively)

3.3.4 Biological activity of hVEGF 165

The biological activity of hVEGF165 secreted from hVEGF165-myoblasts was assessed using HUVEC proliferation and Thymidine [H3] incorporation assays Starting from 1x105, HUVEC showed significantly higher proliferation rate after culturing with supernatant from hVEGF165-myoblasts after 4 days of observation (2.25 ± 0.20 x 105,

p<0.01) as compared with supernatants from non-transduced or Ad-null transduced

myoblasts which showed poor rate of proliferation (1.28 ± 0.09 x 105 and 1.51 ± 0.05 x 105

respectively) (Figure 11a) Inclusion of anti- hVEGF165 antibody inhibited this effect

Similarly, thymidine [H3] incorporation assay showed that scintillation counts/ 10 minutes for HUVEC cultured with supernatant from hVEGF165-myoblasts was highest (4223.6 ± 301.4 counts, p<0.01) compared with supernatants from non-transduced or Ad-

Supernatant Cell lysate

hVEGF165

Figure 9: Western blot analysis of supernatant and lysate from Ad-VEGF165 transduced myoblasts for hVEGF165 expression in vitro

0 5 10 15 20 25 30 35 40 45

Non-Figure 10: Expression of hVEGF165 from Ad-VEGF165 transduced myoblasts as a function

of time in vitro The cells continuously secreted hVEGF165 for up to 30 days post transduction

0 1 2 3

(* vs any other samples: p<0.01)

Figure 11a & b: Assessment of biological activity of hVEGF165 from Ad-VEGF165

transduced myoblasts (a) HUVEC proliferation assay and (b) Thymidine [H3] incorporation assay

A= Fresh Super-medium

B= Ad-null myoblast supernatant

C= Non-transduced myoblast supernatant

D= hVEGF165–myoblast supernatant

E= hVEGF165–myoblast supernatant + anti-VEGF165 antibody

The incorporation of Thymidine [H3] was inhibited and the scintillation count declined to (2379.0 ± 231.8 counts) when anti-VEGF165 antibody was included in the growth well

3.4 Skeletal myoblasts transduction with Ad-Ang-1

As the construct structure of adenoviral vectors of Ad-VEGF165 and Ad-Ang-1 were the same, similar transduction procedure as that of Ad-VEGF165 was used for Ad-Ang-1 transduction with myoblasts The transduction was carried out at a ratio of 1000 PFU/ myoblast, 8 hours exposure time at an interval of 24 hours on three consecutive days Immunostaining of Ang-1 myoblasts revealed high transduction efficiency DAB substrate detection system and fluorescent immunostaining revealed that > 95% transduced myoblasts expressed Ang-1 (Figure 12a & d) as detected by applying anti-Ang-1 antibody, using Ad-null transduced myoblasts as negative controls (Figure 12 b & e)

RT-PCR complied with the findings of immunochemical staining The presence of mRNA encoding for Ang-1 revealed that Ad-Ang-1 transduced myoblasts had a higher level of Ang-1 gene expression as compared with non-transduced and Ad-null transduced myoblasts The peak expression was at day 8 after transduction and decreased with time (Figure 13)

Western blot analysis of supernatants and cell lysates, from Ad-Ang-1 transduced myoblasts, showed the presence of Ang-1 using anti-Ang-1 antibody (Figure 14) The molecular weight of secreted Ang-1 was 70 kDa The concentration of Ang-1 in supernatant was much higher than that in cell lysate thus suggesting that Ang-1 was

using (b) Ad-null myoblasts as a negative control (c) Typical Ad-Ang-1 transduced

myoblasts expressing Ang-1 in vitro (d) Fluorescent immunostaining of skeletal myoblasts for Ang-1 expression as red fluorescence (TRITC) in vitro using (e) Ad-null

transduced myoblasts as a negative control Cells of pictures (a) & (c) were counter stained with methyl green and cells of pictures (d) & (e) were counter-stained with DAPI (blue fluorescence) to visualize nuclei (Magnification a, b, d & e= 300x; c= oil

Figure 13: RT-PCR of Ad-Ang-1 transduced myoblasts for Ang-1 expression in vitro

Lane 1 = DNA ladder

Lane 2 & 3 = Non-transduced myoblasts

Lane 4 & 5 = Ad-null transduced myoblasts

Lane 6~ 13 = Ad-Ang-1transduced myoblasts at 1, 8, 18 and 30

days after transduction

Standard Ang-1 myoblast Ad-null myoblast

Ang-1 supernatant cell lysate supernatantAng-1

Figure 14 Western blot analysis of supernatant and cell lysate from Ad-Ang-1 transduced

myoblasts for Ang-1 expression

3.5 Skeletal myoblast transduction with Ad-Bic

Skeletal myoblasts transduced with Bic-myoblasts were characterized in vitro for co-expression of hVEGF165 and Ang-1

3.5.1 Optimization of transduction condition

The optimization of transduction conditions was carried out as described before for hVEGF165 and Ang-1 Expression efficiency of hVEGF165 was assessed by VEGF ELISA Similarly, a dose-dependent relation between the viral particle number and skeletal myoblast number was observed With the increase of the viral particle concentration, the transduction efficiency also increased (Figure 15a) However, the highest transduction efficiency of skeletal myoblast transduction was achieved at a ratio of 1000 PFU/ myoblast Again, the time of exposure of skeletal myobalsts to Ad-Bic vector showed a direct relation with transduction efficiency (Figure 15b) However, at a higher viral titer (>1000 PFU) or a longer time (>8 hours) exposure, ill effects were observed The optimum transduction efficiency was achieved at a ratio of 1000 PFU/ myoblast when transduction was carried out for 8 hours exposure on three consecutive days

3.5.2 Qualitative assessment of hVEGF 165 expression from Bic-myoblasts

Dual fluorescent immunostaining of Ad-Bic transduced myoblasts revealed that more than 95% Bic-myoblasts concurrently expressed hVEGF165 and Ang-1 (Figure 16) RT-PCR showed that mRNA encoding for hVEGF165 and Ang-1 was detected by hVEGF165 and Ang-1 specific primers Ad-Bic transduced myoblasts had higher levels of hVEGF165 and Ang-1 gene expression as compared with non-transduced and Ad-null transduced myoblasts The peak level was achieved at day 8 after transduction and decreased by day 30 after transduction (Figure 17)

0 500 1000 1500 2000

PBS transduced myoblast

Non-Ad-null myoblast

Figure 15 a & b: Optimization of conditions for skeletal myoblast transduction with

Ad-Bic based on hVEGF165 expression (a) Efficiency of skeletal myoblast transduction as a function of Ad-Bic titer (b) Expression of hVEGF165 from Bic-myoblasts as a function of transduction time Transduction was repeated three times

Figure 16a-d: Dual fluorescent immunostaining of Ad-Bic transduced myoblasts for

hVEGF165 and Ang-1 expression (a) hVEGF165 was visualized as green fluorescence

(FITC) and (b) Ang-1 was visualized as red fluorescence (TRITC) Pictures (a) and (b) were merged to show (c) the concurrent expression of hVEGF165 and Ang-1 (d) Ad-null

transduced myoblasts were used as a negative control Cells were counter stained with DAPI (blue fluorescence) to show nuclei (Magnification = oil immersion)

1 day 8 days 18 days 30 days

Lane 1 = DNA ladder

Lane 2 & 3 = Non-transduced myoblasts

Lane 4 & 5 = Ad-null transduced myoblasts

Lane 6 ~13 = Ad-Bic transduced myoblasts at 1, 8, 18 and 30 days

after transduction

Western blot analysis of supernatant and cell lysate from Ad-Bic transduced myoblasts showed the presence of both hVEGF165 and Ang-1 in the supernatant and cell lysate from Bic-myoblasts (Figure 18) This demonstrated that hVEGF165 and Ang-1 were actively expressed and secreted into the supernatant The molecular weights of hVEGF165and Ang-1 were 42 kDa and 70 kDa respectively

3.5.3 Quantitative assessment of hVEGF 165 expressed by Bic-myoblasts

ELISA for hVEGF165 revealed that Ad-Bic transduced myoblasts under optimum transduction conditions continuously secreted hVEGF165 for up to 30 day (12± 0.6 ng/ ml) with peak level at day 8 (32± 4 ng/ml) The expression continued to decline in a time related manner until day 30 of observation (Figure 19)

3.5.4 Biological activity assessment of hVEGF 165 and Ang-1 secreted by Bic-myoblasts

The biological activity of hVEGF165 and Ang-1 secreted from Ad-Bic transduced skeletal myoblasts was assessed using HUVEC proliferation and thymidine [H3] incorporation assays Starting from 2.5 x104/ well, HUVEC cultured with supernatant from Bic-myoblasts showed highest proliferation rate (5.04± 0.2x104, p<0.01 vs any other

sample) after 4 days’ co-culture compared with supernatants from non-transduced and null transduced myoblasts (2.48± 0.19x104 and 2.38± 0.24 x104 respectively) (Figure 20a) However, cell proliferation rate was reduced to 3.38± 0.24 x104 (p<0.01 vs any other

Ad-sample, except Bic-myoblast supernatant) when anti-Ang-1 antibody alone was added The proliferation of HUVEC was totally inhibited when anti-VEGF165 antibody alone or combining with Ang-1 antibody was applied (2.11± 0.12x104 and 1.69± 0.19x104 respectively)

Standard Bic- myoblast Ad-null myoblast VEGF supernatant cell lysate supernatant

38kDa

a

Ang-1 supernatant supernatant cell lysate

70kDa

b

Standard Standard Bic- myoblast Ad-null myoblast

Ang-1 VEGF supernatant cell lysate supernatant

0 5 10 15 20 25 30 35 40

Non-Figure 19: Expression of hVEGF165 from Ad-Bic transduced myoblasts as a function of time

0 2 4 6

* vs any other sample at day-4: p<0.01;

# vs any other sample at day-4, except sample E: p<0.01.

Figure 20a & b Biological activity assessment of hVEGF165 and Ang-1 secreted from

Bic-myoblasts using (a) HUVEC proliferation and (b) Thymidine [H3] incorporation assays

A = Fresh Super-medium without Thymidine [H3]

B = Fresh Super-medium

C = Ad-null myoblast supernatant

D = Non-transduced myoblast supernatant

E = Bic –myoblast supernatant

F = Bic–myoblast supernatant + anti-VEGF165 antibody

G = Bic–myoblast supernatant +anti-Ang-1 antibody

H = Bic–myoblast supernatant + anti-VEGF165 and anti-Ang-1 antibodies

The thymidine [H3] incorporation assay revealed that HUVEC cultured with supernatant from Bic-myoblasts incorporated the highest level of Thymidine [H3] (3360.58± 115.05 counts) (p<0.01 vs any other sample) after 4 days’ co-culture as compared with supernatants from non-transduced and Ad-null transduced myoblasts (812.6± 82.1 counts and 854± 54.4 counts respectively) (Figure 20b) However, the scintillation counts reduced to 1869.46± 28.55 (vs any other sample, except Bic-myoblast

supernatant: p<0.01) when anti-Ang-1 antibody alone was added The incorporation of

thymidine [H3] by HUVEC was inhibited when anti-VEGF165 antibody alone or together with anti-Ang-1 antibody was applied (776.96± 39.55 and 694.21± 46 respectively) Capillary-like tubular structures were formed when HUVEC were co-cultured with Bic-myoblasts at day 6 after co-culturing (Figure 21a) However, less capillary-like tubular structures were observed when either anti-VEGF165 or anti-Ang-1 antibodies were supplemented into culture medium (Figure 21b & c) Moreover, the capillary-like tubular structure formation was totally blocked when anti-VEGF and anti-Ang-1 antibodies were co-administrated (Figure 21d)

3.6 Skeletal myoblast labeling with nLac-z reporter gene and BrdU

The retroviral vector titer estimated by using NIH 3T3 fibroblasts was ~107 CFU/

ml No replication-competent retroviral vector was found The expression of nLac-z

reporter gene was located in the nucleus (Figure 22a) About 75~80 % of total skeletal

myoblasts were transduced with nLac-z reporter gene using a multiple transduction procedure The optimized procedure for nLac-z reporter gene transduction involved three times transduction of the myoblasts with retrovirus carrying nLac-z reporter gene with

nuclear localization signal Transduction was carried out for 8 hours on three consecutive days (Figure 22b) BrdU staining demonstrated that >95% of labeled skeletal myoblasts

Figure 21a-d: Capillary-like tubular structure formation (a) Capillary-like tubular

structures were formed when HUVEC were co-cultured with Bic-myoblasts Less capillary-like tubular structure was formed when HUVEC was co-cultured with Bic-

myoblasts supplemented with either (b) anti-VEGF165 antibody or (c) anti-Ang-1 antibody

(d) No capillary-like tubular structure was formed when HUVEC were co-cultured with

Bic-myoblasts supplemented with both anti-VEGF165 and Anti-Ang-1 antibodies (Magnification = 100x)

b

a

c

Figure 22a-c: Histochemistry for nLac-z expression in (a) FLY-A4 packaging cells and

(b) human skeletal myoblasts The figures show nuclear localized green color signal after

X-gal staining (c) Immunostaining of human skeletal myoblasts for BrdU localization

The antigen-antibody reaction was visualized by alkaline phosphatase using nitroblue tetrazolium substrate (magnification: a & b= 100x, c= 200x)

3.7 In vivo animal studies of rat heart model

3.7.1 Rat model of cryoinjured heart

A rat heart model of cryoinjury was successfully developed in 53 female Wistar rats Out of 53 rats, 36 animals survived full length of the experiment Another 12 rats were used as normal control ( without any pharmacological or surgical interventions)

3.7.2 Infarction of rat heart

The repeated cycles of freezing and thaw using a liquid nitrogen cooled probe on the left ventricular wall yielded a uniform injury with a diameter of 8 mm (Figure 23a & b) The injury was demonstrated by hematoxylin/ eosin staining at 6 weeks after the development of rat heart model (Figure 23c) A scar was formed in the infarct region in the control animals treated with basal DMEM without skeletal myoblast therapy as visualized by Masson Trichrome staining (Figure 23d)

3.7.3 Survival of skeletal myoblasts in rat heart

X-gal staining for nLac-z expression showed survival of human skeletal myoblasts

in rat heart at 2 days, 2 and 6 weeks after myoblast transplantation (Figure 24a-c) BrdU staining further confirmed the survival of skeletal myoblasts in rat heart (Figure 24 d) The positively stained skeletal myoblasts also showed survival in the infarct and peri- infarct areas (Figure 24b & d) PCR analysis of rat heart tissue for human Y-chromosome detection at 6 weeks after myoblast transplantation reconfirmed this (Figure 25)

3.7.4 RT-PCR analysis for hVEGF 165 expression in rat heart

RT-PCR analysis of rat heart tissue for hVEGF165 expression revealed that hVEGF165-myoblast secreted hVEGF165 for up to 4 weeks of observation after cell transplantation in rat heart (Figure 26) The expression level of hVEGF165 decreased in a time related manner The expression was strongest at 1 week, declined at 4 weeks and

d

c

Figure 23a-d: Rat heart model of cryoinjury was developed by applying (a) liquid

nitrogen cooled probe (b) to achieve a uniform injury size on the left ventricular wall (c)

Hematoxylin/ Eosin staining of cryoinjured rat heart tissue showing injury and the scar was

visualized by (d) Masson Trichome staining of cryoinjured rat heart tissue at 6 weeks after

creating the model (Magnification: c= 100x, d= 400x)

Figure 24a-d: Survival of human skeletal myoblasts in rat heart X-gal staining for nLac-z

expression at (a) 2 days (b) 2 weeks and (c) 6 weeks after skeletal myoblast

transplantation (d) BrdU staining showed survival of human skeletal myoblasts in infarct

area at 6 weeks after cell transplantation (Magnification: a= 100x, and b, c & d= 200x)

(Green = nLac-z expression; purple=BrdU)

1 2 3 4 5 6 7 8

Y Chromosome

Rat GAPDHa

Figure 25: PCR of rat heart tissue at 6 weeks after skeletal myoblast transplantation for

detection of human Y-chromosome in rat heart

Lane 1 = DNA ladder

Lane 2 = human Y chromosome from male human skeletal

myoblasts as a positive control

Lane 3 & 4 = DMEM injected rat heart

Lane 5 & 6 = Non-transduced myoblast transplanted rat heart

Lane 7 & 8 = hVEGF165-myoblast transplanted rat heart

hVEGFB165B-myoblast transplanted at

1 week 4 weeks 6 weeks

1 2 3 4 5 6 7 8 9 10 11 12

Rat GAPDHb

hVEGFB 165

Figure 26: RT-PCR for hVEGF165 expression in rat heart

Lane 1 = DNA ladder

Lane 2 = Ad-VEGF165 transduced myoblasts as a positive controlLane 3 & 4 = DMEM injected rat heart

Lane 5 & 6 = Non-transduced myoblast transplanted rat heart

Lane 7 ~ 12 = hVEGF165-myoblast transplanted rat heart at 1, 4 and 6

weeks

3.7.5 Blood vessel density as revealed by dual fluorescent immunostaining

Fluorescent immunostaining for vWF-VIII and SMA expression demonstrated that capillary density (number of blood vessels per microscopic field) at low power magnification (100x) in hVEGF165-myoblast group was 31.25± 1.82 and 24.63± 0.92 (Figure 27& 28a) These were significantly higher than those of DMEM group (13.29±

1.0, p<0.001; 9.71± 0.81, p<0.001), non-transduced myoblast group (16.50± 1.43, p<0.001; 14.5± 1.34, p<0.001) and normal rats (20.17 ± 1.61, p<0.001; 17.25± 1.17, p<0.001) The blood vessel density of DMEM group was also significantly lower than that

of normal rat group (p=0.004 and p<0.001, respectively) NO significant difference was

observed between normal group and non-transduced myoblast group

Superimposition of images taken for the same microscopic fields for vWF-VIII and SMA showed some of the blood vessels were simple tubular structures of the endothelial cells without smooth muscle coating (Figure 27) Considering the presence of smooth muscle as an indicator for the maturity of the blood vessel, the mature blood vessel index was highest in non-transduced myoblast transplanted group (87.62±1.64%, p=0.017), followed by normal rat group (85.59± 3.27%, p=0.018) and hVEGF165-myoblast transplanted group (79.61± 2.28%, p=0.269) as compared with DMEM injected group (73.93± 4.8%) (Figure 28b)

3.7.6 Improvement of rat heart function

Echo revealed impaired LVEF in control animals treated with DMEM LVEF decreased to 81.8± 3.3% at 6 weeks after DMEM treatment (Figure 29a) However, LVEF

Smooth muscle actin merged vWF-VIII

hVEGF 165 -myoblast group

Figure 27a-l: Visualization of blood vessel in rat heart at low power magnification (100x)

at 6 weeks after cell transplantation Double fluorescent immunostaining for vWF-VIII

0 10 20 30 40

Normal group DM EM group Non-transduced

myoblast group

myoblast group

Normal group DM EM group Non-transduced

myoblast group

myoblast group

Figure 28a-b: Blood vessel density and mature blood vessel index of rat heart (a) Rat

blood vessel density at per low power microscopic field (100x) based on vWF-VIII and

SMA fluorescent immunostaining (b) Mature blood vessel index

Normal group DM EM

Group

transducedmyoblastGroup

Non-myoblast Group

a

020406080100

Normal group DM EM

Group

transducedmyoblastGroup

Non-myoblast Group

Similar improvement was seen in LVFS of hVEGF165-myoblast and

non-transduced myoblast groups, showing 76.13± 2.15% (p=0.011) and 75.0± 3.75% (p=0.024) as compared with DMEM animals (55.1± 7.18%) (Figure 29b)

The left ventricular heart function of non-transduced myoblast and hVEGF165myoblast groups were nearly recovered to normal level (normal group: LVEF= 96.8± 2.36%, LVFS= 81± 7.69%) No significant difference in LVEF and LVFS was observed between hVEGF165-myoblast and non-transduced myoblast groups (Figure 29a & b)

-3.8 In vivo animal studies of pig heart model

Sixty three pigs were used in this study Among these, 6 pigs served as a normal baseline control (normal group without any pharmacological or surgical interventions) Myocardial infarction model was developed in 57 pigs by coronary artery ligation, 7 of which died during the development of the model due to refractory ventricular fibrillation Two pigs died during the second operation (meant for cell transplantation) due to refractory ventricular fibrillation before cell transplantation The remaining 48 pigs survived the full length of experiment without any complications

The pig heart model of chronic infarction was successfully developed by ligation of marginal obtuse branches of LCx coronary artery and confirmed by angiography (Figure 30) and electrocardiography performed at pre- (Figure 31a) and post (Figure 31b) ligation The animals were maintained on a daily dose of 5 mg/ Kg of Cyc-A starting from 5 days prior to and until 6 weeks after myoblast transplantation Electrocardiography of pig heart

Figure 30a-d: Development of pig heart model of myocardial infarction (a) First marginal

obtuse of LCx coronary artery was selected and (b) ligated twice to ensure the successful occlusion of the blood vessel Coronary angiography taken (c) before and (d) after ligation

confirmed the successful occlusion of the blood vessel (Black arrows: marginal obtuse

coronary artery before ligation; yellow arrows: the same coronary artery after ligation)

Figure 31a-e: Electrocardiography of pig heart ECG was recorded at (a) before coronary

artery ligation, (b) after coronary artery ligation, (c) before skeletal myoblast transplantation, (d) during myoblast transplantation and (e) 6 weeks after skeletal myoblast

transplantation (speed= 25 mm/ s)