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Angiogenic potential of endothelial progenitor cells and embryonic stem cells.

Rae PC, Kelly RD, Egginton S, St John JC - (2011)

Bottom Line: We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, UK. justin.stjohn@monash.edu.

ABSTRACT

Background: Endothelial progenitor cells (EPCs) are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC) tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).

Results: The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.

Conclusions: We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.

No MeSH data available.


Related in: MedlinePlus

Characterization of EPCs and ECs. qPCR of (A) VEGFR2, (B) VE-cadherin and (C) CD31 expression at increasing confluency, relative to 60% confluent ECs (mean ± SEM; n = 3). Columns with different letters are significantly different (P < 0.05). ICC of VEGFR2, CD133 and CD34 in (D) EPCs and (E) ECs. Scale bar = 30 μm. (F) Uptake of DiI-ac-LDL (red) and staining with lectin (green). Nuclear staining (blue) with DAPI-containing mounting medium.
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Figure 1: Characterization of EPCs and ECs. qPCR of (A) VEGFR2, (B) VE-cadherin and (C) CD31 expression at increasing confluency, relative to 60% confluent ECs (mean ± SEM; n = 3). Columns with different letters are significantly different (P < 0.05). ICC of VEGFR2, CD133 and CD34 in (D) EPCs and (E) ECs. Scale bar = 30 μm. (F) Uptake of DiI-ac-LDL (red) and staining with lectin (green). Nuclear staining (blue) with DAPI-containing mounting medium.

Mentions: Highly-confluent cells, including the endothelium, demonstrate a reduced response to specific growth factors and proliferative signals such as VEGF [31,32] and contact inhibition is thus implicated as an important endothelial anti-proliferative mechanism. Hence, to determine the effect of contact inhibition on endothelial-specific expression, we analysed the patterns of expression of three key endothelial markers, VEGFR2, VE-cadherin and CD31 in MFLM-4 EPCs and MCEC-1 ECs cultured at 60%, 80% and 100% confluency. Both cell types expressed all three markers at each stage of confluency, though with significant differences whereby increased confluency, and thus contact inhibition, resulted in significant overall decreases in expression (P < 0.01). To this extent, the relative expression of VEGFR2 was significantly greater at 60% and 80% confluency in EPCs than ECs (P < 0.05; Figure 1a). Expression in EPCs increased, though non-significantly, as culture confluency increased from 60% to 80%, whilst no difference in expression was observed between 60% and 80% confluent ECs. Relative expression of VE-cadherin was significantly higher in 60% confluent ECs than in the other cells analyzed (P < 0.05; Figure 1b) and decreased with confluency. VE-cadherin expression in EPCs increased between 60% and 80% confluency. Expression of CD31 was greatest in 60% confluent EPCs but reduced as EPCs became 80% confluent (Figure 1c). CD31 expression was also not significantly different between 60% and 80% confluent ECs. Through ICC staining, we were able to confirm expression of VEGFR2, CD133 and CD34 proteins in EPCs (Figure 1d) and ECs (Figure 1e) with little evidence for change with degree of confluency.


Angiogenic potential of endothelial progenitor cells and embryonic stem cells.

Rae PC, Kelly RD, Egginton S, St John JC - (2011)

Characterization of EPCs and ECs. qPCR of (A) VEGFR2, (B) VE-cadherin and (C) CD31 expression at increasing confluency, relative to 60% confluent ECs (mean ± SEM; n = 3). Columns with different letters are significantly different (P < 0.05). ICC of VEGFR2, CD133 and CD34 in (D) EPCs and (E) ECs. Scale bar = 30 μm. (F) Uptake of DiI-ac-LDL (red) and staining with lectin (green). Nuclear staining (blue) with DAPI-containing mounting medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108917&req=5

Figure 1: Characterization of EPCs and ECs. qPCR of (A) VEGFR2, (B) VE-cadherin and (C) CD31 expression at increasing confluency, relative to 60% confluent ECs (mean ± SEM; n = 3). Columns with different letters are significantly different (P < 0.05). ICC of VEGFR2, CD133 and CD34 in (D) EPCs and (E) ECs. Scale bar = 30 μm. (F) Uptake of DiI-ac-LDL (red) and staining with lectin (green). Nuclear staining (blue) with DAPI-containing mounting medium.
Mentions: Highly-confluent cells, including the endothelium, demonstrate a reduced response to specific growth factors and proliferative signals such as VEGF [31,32] and contact inhibition is thus implicated as an important endothelial anti-proliferative mechanism. Hence, to determine the effect of contact inhibition on endothelial-specific expression, we analysed the patterns of expression of three key endothelial markers, VEGFR2, VE-cadherin and CD31 in MFLM-4 EPCs and MCEC-1 ECs cultured at 60%, 80% and 100% confluency. Both cell types expressed all three markers at each stage of confluency, though with significant differences whereby increased confluency, and thus contact inhibition, resulted in significant overall decreases in expression (P < 0.01). To this extent, the relative expression of VEGFR2 was significantly greater at 60% and 80% confluency in EPCs than ECs (P < 0.05; Figure 1a). Expression in EPCs increased, though non-significantly, as culture confluency increased from 60% to 80%, whilst no difference in expression was observed between 60% and 80% confluent ECs. Relative expression of VE-cadherin was significantly higher in 60% confluent ECs than in the other cells analyzed (P < 0.05; Figure 1b) and decreased with confluency. VE-cadherin expression in EPCs increased between 60% and 80% confluency. Expression of CD31 was greatest in 60% confluent EPCs but reduced as EPCs became 80% confluent (Figure 1c). CD31 expression was also not significantly different between 60% and 80% confluent ECs. Through ICC staining, we were able to confirm expression of VEGFR2, CD133 and CD34 proteins in EPCs (Figure 1d) and ECs (Figure 1e) with little evidence for change with degree of confluency.

Bottom Line: We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, UK. justin.stjohn@monash.edu.

ABSTRACT

Background: Endothelial progenitor cells (EPCs) are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC) tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs) by direct differentiation using EC-conditioned medium (ECCM).

Results: The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs.

Conclusions: We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.

No MeSH data available.


Related in: MedlinePlus