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Enhancing the function of CD34(+) cells by targeting plasminogen activator inhibitor-1.

Hazra S, Stepps V, Bhatwadekar AD, Caballero S, Boulton ME, Higgins PJ, Nikonova EV, Pepine CJ, Thut C, Finney EM, Stone DJ, Bartelmez SH, Grant MB - PLoS ONE (2013)

Bottom Line: Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications.To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO.We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34(+) cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+) cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34(+) cells. To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

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Inhibition of PAI-1 using miR-146a mimic.(a) Over expression of miR-146a mimic in CD34+ cells. CD34+ cells transfected with miR-146a mimic followed by incubation for 24hrs. RNA isolates were prepared from cell pellets followed by Real-time RT-PCR for miR-146a expression normalized to RNU6B. (b) Transfected miR-146a mimic decreases PAI-1 mRNA expression in CD34+ cells. Real-time RT-PCR for PAI-1 expression was normalized to β-actin. (c) Reduction of secreted PAI-1 in the conditioned media from non-diabetic CD34+ cells. (d) Reduced secreted PAI-1 in the conditioned media from diabetic CD34+ cells.
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pone-0079067-g004: Inhibition of PAI-1 using miR-146a mimic.(a) Over expression of miR-146a mimic in CD34+ cells. CD34+ cells transfected with miR-146a mimic followed by incubation for 24hrs. RNA isolates were prepared from cell pellets followed by Real-time RT-PCR for miR-146a expression normalized to RNU6B. (b) Transfected miR-146a mimic decreases PAI-1 mRNA expression in CD34+ cells. Real-time RT-PCR for PAI-1 expression was normalized to β-actin. (c) Reduction of secreted PAI-1 in the conditioned media from non-diabetic CD34+ cells. (d) Reduced secreted PAI-1 in the conditioned media from diabetic CD34+ cells.

Mentions: Three separate approaches were used to reduce PAI-1 in CD34+ cells; PAI-1 siRNA, lentivirus expressing PAI-1 shRNA (Figure 3) and over-expressing miR-146a mimic (Figure 4). All three approaches were efficient in reducing PAI-1 mRNA. In the presence of growth factors, inhibition of PAI-1 promoted cell growth in CD34+ cells of diabetic and non-diabetic origin (Figure 5a, b). The growth of CD34+ cells of non-diabetic origin (Figure 5a) following PAI-1 inhibition (solid line) was greater than cells treated with lenti shRNA control (dotted line). In contrast, the growth of CD34+ cells of diabetic origin did not improve (dotted line) even in the presence of growth factors; however, inhibition of PAI-1 remarkably increased the rate of growth of the cells of diabetic origin (solid line) to the level of non-diabetic cells (Figure 5b).


Enhancing the function of CD34(+) cells by targeting plasminogen activator inhibitor-1.

Hazra S, Stepps V, Bhatwadekar AD, Caballero S, Boulton ME, Higgins PJ, Nikonova EV, Pepine CJ, Thut C, Finney EM, Stone DJ, Bartelmez SH, Grant MB - PLoS ONE (2013)

Inhibition of PAI-1 using miR-146a mimic.(a) Over expression of miR-146a mimic in CD34+ cells. CD34+ cells transfected with miR-146a mimic followed by incubation for 24hrs. RNA isolates were prepared from cell pellets followed by Real-time RT-PCR for miR-146a expression normalized to RNU6B. (b) Transfected miR-146a mimic decreases PAI-1 mRNA expression in CD34+ cells. Real-time RT-PCR for PAI-1 expression was normalized to β-actin. (c) Reduction of secreted PAI-1 in the conditioned media from non-diabetic CD34+ cells. (d) Reduced secreted PAI-1 in the conditioned media from diabetic CD34+ cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815099&req=5

pone-0079067-g004: Inhibition of PAI-1 using miR-146a mimic.(a) Over expression of miR-146a mimic in CD34+ cells. CD34+ cells transfected with miR-146a mimic followed by incubation for 24hrs. RNA isolates were prepared from cell pellets followed by Real-time RT-PCR for miR-146a expression normalized to RNU6B. (b) Transfected miR-146a mimic decreases PAI-1 mRNA expression in CD34+ cells. Real-time RT-PCR for PAI-1 expression was normalized to β-actin. (c) Reduction of secreted PAI-1 in the conditioned media from non-diabetic CD34+ cells. (d) Reduced secreted PAI-1 in the conditioned media from diabetic CD34+ cells.
Mentions: Three separate approaches were used to reduce PAI-1 in CD34+ cells; PAI-1 siRNA, lentivirus expressing PAI-1 shRNA (Figure 3) and over-expressing miR-146a mimic (Figure 4). All three approaches were efficient in reducing PAI-1 mRNA. In the presence of growth factors, inhibition of PAI-1 promoted cell growth in CD34+ cells of diabetic and non-diabetic origin (Figure 5a, b). The growth of CD34+ cells of non-diabetic origin (Figure 5a) following PAI-1 inhibition (solid line) was greater than cells treated with lenti shRNA control (dotted line). In contrast, the growth of CD34+ cells of diabetic origin did not improve (dotted line) even in the presence of growth factors; however, inhibition of PAI-1 remarkably increased the rate of growth of the cells of diabetic origin (solid line) to the level of non-diabetic cells (Figure 5b).

Bottom Line: Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications.To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO.We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34(+) cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34(+) cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34(+) cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34(+) cells. To reduce PAI-1 in human CD34(+) cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34(+) cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

Show MeSH
Related in: MedlinePlus