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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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PAI-1 inhibition with PMO increases diabetic CD34+ cell homing to and association with vasculature in an acute I/R model.CD34+ cells isolated from diabetic (n=8) or age- and sex-matched normal (n=4) donors were exposed to either scrambled (SCR) or PAI-1-specific PMO for 16hr prior to intravitreous injection 7 days post ischemic insult. Eyes were harvested 2 days later and processed for immunofluorescence and data was collected using laser scanning confocal microscopy. Panels (a-d) are typical fields that show calculated co-localization of CD34+ cells with retinal vasculature using Intensity Correlation Analysis (Mander’s coefiicient) and given false color to indicate the degree of co-localization probability (warmer colors = higher probability). Scale bar in (b) also applies to (a) and is 100 mm. Scale bar in (d) also applies to (c) and is 150 mm. (e) represents co-localization efficiency. Arrows indicate exogenously added cells that have not co-localized to vasculature (best seen in Panel C), while arrowheads (Panel A) indicate areas where exogenously added cells co-localize strongly (warm colors) with vessels. * = p<0.05 vs. non-diabetic cells with the same pre-treatment. † = p<0.05 versus SCR PMO with diabetic cells.
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pone-0079067-g007: PAI-1 inhibition with PMO increases diabetic CD34+ cell homing to and association with vasculature in an acute I/R model.CD34+ cells isolated from diabetic (n=8) or age- and sex-matched normal (n=4) donors were exposed to either scrambled (SCR) or PAI-1-specific PMO for 16hr prior to intravitreous injection 7 days post ischemic insult. Eyes were harvested 2 days later and processed for immunofluorescence and data was collected using laser scanning confocal microscopy. Panels (a-d) are typical fields that show calculated co-localization of CD34+ cells with retinal vasculature using Intensity Correlation Analysis (Mander’s coefiicient) and given false color to indicate the degree of co-localization probability (warmer colors = higher probability). Scale bar in (b) also applies to (a) and is 100 mm. Scale bar in (d) also applies to (c) and is 150 mm. (e) represents co-localization efficiency. Arrows indicate exogenously added cells that have not co-localized to vasculature (best seen in Panel C), while arrowheads (Panel A) indicate areas where exogenously added cells co-localize strongly (warm colors) with vessels. * = p<0.05 vs. non-diabetic cells with the same pre-treatment. † = p<0.05 versus SCR PMO with diabetic cells.

Mentions: The in vivo vasoreparative function of PAI-1 PMO modified CD34+ cells was evaluated using a mouse model of I/R injury that recapitulates many features of diabetic retinopathy, including the presence of acellular capillaries [35]. Cells were injected intravitreally into injured eyes, and the homing of cells to areas of injury, a direct indicator of the in vivo migratory prowess of these cells, was expressed as percent of the total vascular area. Previously, we showed that cells of diabetic origin display markedly reduced homing to areas of injury. Cells of diabetic origin form aggregates on the surface of the vitreous and do not associate with the retinal vasculature [29,36]. In the I/R model of acute retinal vascular injury, between 60-70% of detected CD34+ cells from healthy donors home to and associate with vasculature [29,36]. No difference was measured in association of CD34+ cells of non-diabetic origin with vasculature in cells pre-treated with either scrambled PMO or cells pretreated with PAI-1 PMO. By contrast, CD34+ cells from diabetic donors pre-treated with scrambled PMO exhibited poor homing and association with vasculature, with less than 20% of detected cells co-localizing with vessels. In contrast, when these CD34+ cells were treated with PAI-1 PMO they showed a marked increase in co-localization with injured retinal vasculature (Figure 7) (p<0.05).


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)

PAI-1 inhibition with PMO increases diabetic CD34+ cell homing to and association with vasculature in an acute I/R model.CD34+ cells isolated from diabetic (n=8) or age- and sex-matched normal (n=4) donors were exposed to either scrambled (SCR) or PAI-1-specific PMO for 16hr prior to intravitreous injection 7 days post ischemic insult. Eyes were harvested 2 days later and processed for immunofluorescence and data was collected using laser scanning confocal microscopy. Panels (a-d) are typical fields that show calculated co-localization of CD34+ cells with retinal vasculature using Intensity Correlation Analysis (Mander’s coefiicient) and given false color to indicate the degree of co-localization probability (warmer colors = higher probability). Scale bar in (b) also applies to (a) and is 100 mm. Scale bar in (d) also applies to (c) and is 150 mm. (e) represents co-localization efficiency. Arrows indicate exogenously added cells that have not co-localized to vasculature (best seen in Panel C), while arrowheads (Panel A) indicate areas where exogenously added cells co-localize strongly (warm colors) with vessels. * = p<0.05 vs. non-diabetic cells with the same pre-treatment. † = p<0.05 versus SCR PMO with diabetic cells.
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Related In: Results  -  Collection

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pone-0079067-g007: PAI-1 inhibition with PMO increases diabetic CD34+ cell homing to and association with vasculature in an acute I/R model.CD34+ cells isolated from diabetic (n=8) or age- and sex-matched normal (n=4) donors were exposed to either scrambled (SCR) or PAI-1-specific PMO for 16hr prior to intravitreous injection 7 days post ischemic insult. Eyes were harvested 2 days later and processed for immunofluorescence and data was collected using laser scanning confocal microscopy. Panels (a-d) are typical fields that show calculated co-localization of CD34+ cells with retinal vasculature using Intensity Correlation Analysis (Mander’s coefiicient) and given false color to indicate the degree of co-localization probability (warmer colors = higher probability). Scale bar in (b) also applies to (a) and is 100 mm. Scale bar in (d) also applies to (c) and is 150 mm. (e) represents co-localization efficiency. Arrows indicate exogenously added cells that have not co-localized to vasculature (best seen in Panel C), while arrowheads (Panel A) indicate areas where exogenously added cells co-localize strongly (warm colors) with vessels. * = p<0.05 vs. non-diabetic cells with the same pre-treatment. † = p<0.05 versus SCR PMO with diabetic cells.
Mentions: The in vivo vasoreparative function of PAI-1 PMO modified CD34+ cells was evaluated using a mouse model of I/R injury that recapitulates many features of diabetic retinopathy, including the presence of acellular capillaries [35]. Cells were injected intravitreally into injured eyes, and the homing of cells to areas of injury, a direct indicator of the in vivo migratory prowess of these cells, was expressed as percent of the total vascular area. Previously, we showed that cells of diabetic origin display markedly reduced homing to areas of injury. Cells of diabetic origin form aggregates on the surface of the vitreous and do not associate with the retinal vasculature [29,36]. In the I/R model of acute retinal vascular injury, between 60-70% of detected CD34+ cells from healthy donors home to and associate with vasculature [29,36]. No difference was measured in association of CD34+ cells of non-diabetic origin with vasculature in cells pre-treated with either scrambled PMO or cells pretreated with PAI-1 PMO. By contrast, CD34+ cells from diabetic donors pre-treated with scrambled PMO exhibited poor homing and association with vasculature, with less than 20% of detected cells co-localizing with vessels. In contrast, when these CD34+ cells were treated with PAI-1 PMO they showed a marked increase in co-localization with injured retinal vasculature (Figure 7) (p<0.05).

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