Differential Expression of Transforming Growth Factor Beta Receptor 2 (TGFβR2) In Diabetic CD34+ Cells: Implications for Vascular Repair

Abstract 4795

We recently reported that the vascular reparative ability of diabetic CD34+ cells can be restored by transient inhibition of transforming growth factor β1 (TGF-b1) using antisense phosphorodiamidate morpholino oligomers (TGF-β1-PMO) (Bhatwadekar 2010). TGF-β1 mediates its action by binding to TGF-β-R2 receptor homodimers, which then form heterotetrameric complexes with two TGF-βR1 subunits. We investigated whether healthy or diabetic CD34+ cells differentially express TGFβR2 receptor and whether inhibition or knock-out of endogenous TGF-β1 expression would affect the surface expression of TGF-βR2.

TGF-βR2 expression of murine bone marrow (BM) lin^- Sca-1+/− cells from TGF-β1 knockout mice or wild type mice was studied using flow cytometry. Unexpectedly, TGF-β-R2 expression was essentially absent in BM lin^- Sca-1 +and - cells from TGF-β1 knockout mice compared to wild type cells. Because the absence of endogenous and exogenous TGF-β1 resulted in complete down regulation of TGF-βR2 in primitive murine hematopoietic cells, we explored the relationship of endogenous TGF-β1in human mature and immature CD34+ cells in blood, bone marrow and cord blood.

Human CD34+CD45+ CD38+ or - cells from healthy and diabetic individuals were FACS sorted based on surface expression the analysed for TGF-βR2 expression. At the same time, TGF-βR2 and TGF-β1 mRNA expression was determined for sorted populations using quantitative real time RT-PCR.

A comparison of TGF-βR2 surface expression between mature progenitors (lin^- CD34⁺ CD45⁺ CD38⁺) from diabetic and healthy cord blood revealed that TGF-βR2 expression did not differ between these groups. In contrast, TGF-βR2 surface expression in immature cells (CD34⁺ CD45⁺ CD38^-) was very low in diabetic patients compared to healthy controls (p = 0.001).

Next, we inhibited endogenous TGF-β1 in healthy CD34+ cells which resulted in a significant (p<0.01) reduction in TGF-β-R2 mRNA expression. However, in diabetic CD34 cells, TGF β1-PMO treatment abolished TGFβR2 expression.

Our study suggests that healthy and diabetic CD34+ cells differentially regulate TGF-βR2 expression which may be the result of the elevated intracellular and extracellular levels of TGFb1 in diabetic individuals. A reduction in TGF-βR2 expression in diabetic CD34⁺ CD38- cells may physiologically enhance proliferation of the primitive CD34+ CD38- cell compartment, an outcome that may be necessary to keep up with increased demand for CD34+ cells in the diabetic individual.