Sonic hedgehog is a potent chemoattractant for human monocytes: diabetes mellitus inhibits Sonic hedgehog-induced monocyte chemotaxis

Abstract

The aim of the present study was to evaluate the expression of hedgehog (Hh) signaling molecules and the chemotactic activity of Sonic hedgehog (Shh) in monocytes from control (CTR) and diabetic patients with or without coronary artery disease (CAD). Previously several studies demonstrated that exogenous administration of Shh can induce angiogenesis and accelerate repair of ischemic myocardium and skeletal muscles. Blood samples were collected from (1) CTR (n = 25); (2) patients with stable CAD without diabetes mellitus (CAD-DM, n = 10); and (3) with stable CAD with DM (CAD+DM, n = 15). Monocytes were isolated by Percoll gradient and subjected to PCR and chemotaxis analysis. Hh signaling molecules were expressed in human monocytes, and Shh-induced monocyte chemotaxis. Shh-stimulated migration of monocytes from CTR measured 172.5 +/- 90% and a maximal stimulation was observed at Shh concentration of 1 microg/ml. However, Shh failed to induce migration of monocytes from CAD+DM (94.3 +/- 27%, P < 0.001 vs. CTR). The impaired response to Shh was associated with strong transcriptional upregulation of the receptor Ptc, while expression of downstream molecules was not altered. Moreover, Ptc is strongly expressed in macrophages of human aortic atherosclerotic plaque. Thus, Shh is a potent chemoattractant for monocytes and it activates classical signaling pathways related to migration. The Shh signaling was negatively affected by DM which might be involved in the pathogenesis of DM-related complications.

Fig. 1

Expression of Hedgehog signaling components in human monocytes. a RT-PCR analysis of the Shh, Ihh, Dhh, Smo, Ptc, SUFU, Hip, Gli1, Gli2, Gli3, and actin gene expression on human peripheral monocytes and HUVEC (used as a positive control). The results are representative of three independent experiments. b Flow cytometric analysis of Ptc expression on monocytes. Left panel Isotype (negative) control. Right panel Purified monocytes were double-stained with anti-Ptc antibodies and CD14 antibodies as a marker for monocytes

Fig. 2

Both Shh (a) and Ihh (b) induce migration of human monocytes ex vivo in a concentration-dependent fashion. Monocytes were isolated from peripheral blood samples (n = 25) and the chemotactic response was measured using a modified Boyden chamber. Five high-power fields were counted for each sample (primary magnification ×20). Statistical significance was evaluated using the unpaired Student’s t test. The median values are plotted on the graphs. * P ≤ 0.005. c Monocyte migration in the presence (right) or absence (left) of Shh (1 μg/ml). The representative photograph of the stained filter; monocytes are detected on the lower surface of the filter following migration through the pores

Fig. 3

a Cyclopamine (CP) inhibits Shh-induced monocyte chemotaxis. Monocytes were pretreated with CP (10 μM) for 15 min and chemotactic response to Shh (1 μg/ml) was measured using the modified Boyden chamber. Statistical significance was evaluated using the unpaired Student’s t test. The median values are plotted on the graphs; * P < 0.005, n = 11. Both G_i inhibitor PTX (100 ng/ml, b) and PI3K inhibitor LY294002 (10 μM, c) block Shh-induced (1 μg/ml) chemotaxis of peripheral monocytes. The median values are plotted on the graphs; * P < 0.005, n = 11

Fig. 4

a Shh-induced (1 μg/ml) chemotaxis of monocytes isolated from CTR, CAD−DM or CAD+DM patients. The median values are plotted; ns; nonsignificant versus unstimulated control. The median (line), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) are presented. * P < 0.005. The level of Ptc (b), and Ihh (c) gene expression on monocytes isolated from CTR, CAD−DM and CAD+DM patients. RNA isolated from monocytes was subjected to RT-PCR. β-actin cDNA product served as internal standard to normalize Ptc and Ihh. Signal intensity of Ptc and Ihh mRNA bands was quantified by densitometry and compared with the internal standard β-actin

Fig. 5

Expression of Ptc in an advanced, stable human aortic atherosclerotic lesion. Immunohistological analysis demonstrates that approximately 80% of macrophages were positive for both Ptc and CD68 (merge). Brown color indicates positivity. Sections were counterstained with hematoxylin. Magnification for upper panels is ×10 and for lower panels (magnified inserts) is ×40