Introduction
The Hedgehog (Hh) pathway is highly conserved in evolution and Hh proteins are essential for cell fate decisions during development and homeostasis of adult tissues. Three proteins compose the Hh family: Sonic (Shh), Indian (Ihh) and Desert (Dhh) hedgehogs. All Hh proteins bind to a surface receptor complex comprised of two transmembrane proteins Patched (Ptc), a receptor for Hh proteins and Smoothened (Smo), a signal transducer. It has been demonstrated that all Hh members Shh, Ihh and Dhh proteins bind the receptor Ptc with similar affinity, however, they vary with regard to their potency. The rank order of potency was generally Shh ≥ Ihh ≥ Dhh [
27]. The Hh signal is then mediated by the Gli family transcription factors (Gli 1–3), which regulate the expression of different target genes [
10].
There is increasing evidence for the role of Hh signaling in vascularization and neovascularization. Recently, it has been shown that activation of Hh signaling is critical for coronary development and sufficient to promote formation of coronary vessels in the embryonic and adult heart [
13,
22,
29]. Shh gene therapy could promote both angiogenesis and arteriogenesis and protect from ischemic injury in rodent and large animal models [
21]. However, the mechanism how Shh induces neovascularization is still unknown. It has been shown that blood circulating mononuclear cells play an important role in both artheriogenesis and angiogenesis [
15,
32]. They adhere to the arterial wall, infiltrate and stimulate vessel growth in the ischemic tissue by the release of cytokines, growth factors and proteases. Several recent studies have suggested a role of Hh signaling in the control of motility and migration of multiple cell types. Hedgehog proteins act as a chemoattractant on isolated axons, guide neuronal migration during embryonic development [
6], serves as an attractive cue to guide germ cell migration through the embryo to form primitive gonad in
Drosophila [
12], promote the migration of endothelial progenitor cells [
1], rat activated pancreatic stellate cells [
33], embryonic endothelial cells and fibroblasts [
18] as well as optic nerve oligodendrocyte precursors [
24]. However, nothing is known about migration control of immune cells by Hh proteins. Here, we tested the hypothesis whether Shh stimulates monocyte chemotaxis. Our results demonstrate that Shh is a potent chemoattractant for peripheral monocytes and it activates classical signaling pathways related to cellular migration such as G-proteins or PI3K. Moreover, we provide the first piece of evidence that pathological conditions such as diabetes mellitus (DM) significantly impair Shh-induced chemotaxis, which is accompanied by elevated expression levels of Ptc. Thus, these data indicate that the Hh signaling pathway (1) is involved in monocyte biology and (2) is negatively affected by the cardiovascular risk factor DM.
Methods
Study population
This study was performed with the approval of the medical ethics committee of the University Hospital of Maastricht (The Netherlands), and conforms to the principals outlined in the Declaration of Helsinki. All enrolled subjects gave their written informed consent. Three groups of subjects were studied: (1) Patients with stable coronary artery disease without diabetes mellitus (CAD−DM,
n = 10). Stable CAD was defined as history of stable angina pectoris or history of PCI and/or CABG. Patients with acute myocardial infarction and/or recent surgical intervention (less then 6 months) were excluded. All patients had a recent coronary angiogram. (2) CAD patients with diabetes mellitus (CAD+DM,
n = 15). The diagnosis of DM was verified by history of DM, elevated fasting glucose level and/or increased concentration of glycosylated HbA1
c. (3) Control subjects (CTR) without any history or clinical signs of CAD, metabolic disease or chronic illness (
n = 25). Baseline clinical features of CTR, CAD−DM and CAD+DM patients are presented in Table
1. There were no significant differences between three groups with regards to age, gender, BMI, presence of cardiovascular risk factors and medication. CAD+DM patients exhibited higher fasting glucose level compared to CTR (
P = 0.019) and compared to CAD−DM group (
P = 0.048).
Table 1
Clinical characteristics of control subjects and patients
Age (years) | 59.9 ± 12.7 | 63.7 ± 9.9 | 66 ± 11 | 0.579 |
BMI | ND | 26.8 ± 2 | 26.3 ± 5 | |
Male gender, n (%) | 12 (48) | 7 (70) | 9 (60) | 0.470 |
Cardiovascular risk factors |
CAD history (years) | 0 | 10 ± 8 | 9 ± 8 | 0.578 |
Family history of CAD, n (%) | 11 (44) | 7 (70) | 7 (47) | 0.231 |
Hypertension, n (%) | 12 (48) | 4 (40) | 9 (60) | 0.284 |
Smoking, n (%) | 6 (24) | 3 (30) | 6 (40) | 0.607 |
Diabetes, n (%) | 0 (0) | 0 (0) | 15 (100) | |
Duration of DM (years) | 0 | 0 | 8 ± 7 | |
Patients receiving insulin (%) | 0 | 0 | 40 | |
Patients receiving OHD (%) | 0 | 0 | 100 | |
FPG (mmol/l) | 5.5 ± 0.7 | 6.3 ± 0.7 | 8.3 ± 4.6 | 0.048 |
HbA1c (%) | ND | ND | 7.3 ± 0.6 | |
Laboratory parameters |
Cholesterol, total (mmol/l) | 5.9 ± 1.3 | 4.4 ± 0.8 | 4.0 ± 1.4 | 0.283 |
LDL-cholesterol (mmol/l) | 4.0 ± 1.2 | 2.1 ± 0.8 | 2.1 ± 0.9 | 0.16 |
HDL-cholesterol (mmol/l) | 1.8 ± 1.0 | 1.2 ± 0.6 | 1.0 ± 0.3 | 0.127 |
Triglycerides (mmol/l) | 1.8 ± 1.2 | 1.8 ± 0.4 | 2.1 ± 1.1 | 0.638 |
Medication at admission |
Antiaggregatory therapy (antiplatelet drugs) n (%) | 11 (44) | 7 (70) | 7 (47) | 0.231 |
Beta-blockers, n (%) | 16 (64) | 7 (70) | 10 (67) | 0.222 |
ACE-inhibitors/ARB, n (%) | 12 (48) | 6 (60) | 7 (47) | 0.688 |
Statins, n (%) | 12 (48) | 8 (80) | 12 (80) | 0.687 |
Monocyte isolation and migration
Blood samples (100 ml) were collected from all subjects. Monocytes were isolated by Percoll gradient centrifugation based on a previously described protocol [
40]. The purity of isolated monocytes was ≥90% as determined by flow cytometry. Monocytes were subjected to chemotaxis assay using the modified Boyden chamber [
2]. Briefly, different concentrations of Shh or Ihh were placed in the bottom well and monocytes were placed in the top well. Polycarbonate membranes with a pore diameter of 5 μm (Nuclepore) were used. The chambers were incubated at 37°C in the presence of 5% CO
2 for 90 min. Afterwards filters were removed, fixed, stained with Giemsa dye before scraping off cells at the upper side of the filter membrane and five high-power fields were counted for each sample (primary magnification 20×). To differentiate between chemotaxis and chemokinesis, checkerboard analysis was performed by placing various dilutions of Shh in both the lower and upper wells of the modified Boyden chamber. To study the effect of different inhibitors on Shh-induced migration, monocytes were pretreated with a specific inhibitor of Hh signaling, cyclopamine (CP, 10 μM), the PI3K inhibitor, LY294002 (10 μM), or a specific inhibitor of Gα
i/o, pertussis toxin (PTX, 100 ng/ml) for 15 min before the chemotaxis assay.
RT-PCR analysis
Total RNA from monocytes or HUVEC (used as a control) was isolated using RNeasy Protect Mini Kit from QIAGEN. For reverse transcription, 1 μg of total RNA was converted into cDNA by AMV reverse transcriptase at 37°C for 1 h in a 20 μl RT reaction. All the primers used in this study and PCR conditions are listed in Table
2.
Table 2
Primers and conditions used for RT-PCR
Shh | FW: ACT GGG TGT ACT ACG AGT CCA AGG | 63°C, 50 cycles | 211 |
RV: AAA GTG AGG AAG TCG CTG TAG AGC |
Ihh | FW: CTA CGC CCC GCT CAC AAA G | 60°C, 30 cycles | 376 |
RV: GGC AGA GGA GAT GGC AGG AG |
Dhh | FW: ACCAATCTACTGCCCCTGTG | 62°C, 30 cycles | 246 |
RV: GTTGTAGTTGGGCACGAGGT |
Smo | FW: CAG GAC ATG CAC AGC TAC ATC G | 65°C, 30 cycles | 380 |
RV: CCA CAA AGA AGC ACG CAT TGA C |
Ptc | FW: CCA TGT TCC AGT TAA TGA CTC | 55°C, 30 cycles | 462 |
RV: ACA TCA TCC ACA CCA ACA |
SUFU | FW: CCT CCA GAT CGT TGG TGT CT | 55°C, 30 cycles | 195 |
RV: CTG TCT CGA TGC CTT TGT CA |
Gli-1 | FW: GGG ATG ATC CCA CAT CCT CAG TC | 60°C, 50 cycles | 386 |
RV: CTG GAG CAG CCC CCC CAG T |
Gli-2 | FW: ACC GCT GCT CAA AGA GAA TG | 64°C, 50 cycles | 507 |
RV: CCC ACT GCC ACT GAA GTT TTC C |
Gli3 | FW: CCT CAA AGC GGG CCG CCT GC | 64°C, 50 cycles | 406 |
RV: CAG GTT GTT GTT GGA CTG TGT GC |
Flow cytometry analysis
Expression of Ptc on leukocytes was analyzed by flow cytometry. Leukocytes were stained with anti-Ptc primary antibodies (Santa Cruz Biotechnology). Double staining with CD14-FITC (Becton Dickinson) and Ptc/anti-goat-PE antibody was performed to confirm the expression of both proteins on monocytes. The analysis was carried out on a FACSCalibur flow cytometer using the CellQuest software (Becton Dickinson).
Immunohistochemistry
Cross sections of human aortic atherosclerotic plaque were deparaffinized, dehydrated, and permeabilized with 0.05% Tween in citrate buffer, and then blocked with normal horse serum. Further, they were incubated with primary polyclonal antibodies for Ptc (1:25, goat polyclonals; Santa Cruz Biotechnology Inc) or CD68 (1:100, mouse monoclonal; DakoCytomation). A biotinylated horse anti-mouse and anti-goat IgG (1:1,000) was incubated followed by incubation with streptavidin/HRP antibodies and visualized with AEC staining solution. Consecutive sections were stained and the results of Ptc staining and CD68 staining were merged for demonstration of coexpression. The negative controls included substitution of primary antiserum for PBS.
Statistical analysis
Statistical analysis was performed with SPSS12.0.1 statistical software. Categorical variables are presented as percentage of patients and compared by Fisher’s exact test. Data are presented as the mean ± standard deviation (SD). In addition, the medians (in the figure: median as line, 25th–75th percentiles as box, 5th–95th percentiles as whiskers) are given in case of non-normally distributed results. CTR, CAD−DM and CAD+DM patients were compared using a Mann–Whitney U test with Bonferroni correction. Values of P < 0.05 were considered statistically significant.
Discussion
In this study, we present data supporting a role for Shh/Ihh in stimulation of monocyte chemotaxis. We have not used Dhh for our experiments, because up to now there is no evidence that Dhh is expressed in vascular endothelial cell or vascular smooth muscle cells. Our data demonstrate that both Shh and Ihh stimulate monocyte chemotaxis at the optimal concentration for Shh 1 μg/ml and for Ihh 2.5 μg/ml. Chemotaxis induced by Shh was specific and Smo-dependent. Moreover, Shh activates classical intracellular signal transduction pathways related to cellular migration such as G-protein-coupled specific receptor pathways or PI3K.
The Shh concentration, which stimulates a maximum effect, is rather high in comparison with other known chemoattractants such as MCP-1, MIPs and fractalkine [
25]. This may be explained as follows: Hh proteins undergo two posttranslational processing events that result in the covalent addition of cholesterol at the C-terminus [
30] and a long-chain fatty acid on the N-terminal cysteine [
34]. In addition to tethering the protein to the surface of the cell in which it is synthesized, lipid modification greatly stimulates biological activity of the protein [
28,
37]. Recombinant Shh used in these experiments was expressed and purified from
E. coli, where it does not undergo those hydrophobic modifications. Recombinant Shh is therefore less potent that natural modified proteins [
38].
Our current study was not designed to identify the physiological source of Shh that could influence monocyte migration. Nevertheless, it has already been demonstrated that vascular endothelial [
4] and smooth muscle cells [
25] express Shh. It is thus conceivable that Shh expressed in these cells may attract monocytes into a vessel. Moreover, altered expression of Shh has been reported in a variety of different pathological conditions such as tumorgenesis [
14] and ischemic injury [
21]. Expression of both Shh and Ptc was increased in cardiac ischemia. Shh expression was upregulated at the sites of inflammation [
29] and repair [
41].
Recent reports have indicated that abnormalities in monocyte function may be one of the factors responsible for reduced vessel growth and vascular complications in certain patient groups [
39]. Monocytes from diabetic patients show a migration defect [
40], reduced phagocytosis [
20,
26] and increased adhesion to endothelial cells [
9]. The present study demonstrates that monocytes from diabetic patients do not respond to Shh at the most efficient concentration in CTR, i.e. 1 μg/ml. This impaired chemotactic response was associated with a 11-fold increase of Ptc mRNA expression in diabetic monocytes. The increase of Ptc expression could be also observed for some CAD−DM patients; however, the difference in expression was not statistically significant in comparison to control subjects (3 patients from 10 had higher level of Ptc expression compared to control). However, there was no significant difference in mRNA expression between different groups for ligands Ihh and Dhh and downstream Hh signaling components Smo and SUFU. Previously, impaired chemotactic response of monocytes associated with increased surface expression of receptors was observed for patients with DM type 1 [
5]. Similar effect was observed for blood monocytes from epithelial ovarian cancer. Notwithstanding the increased levels of CCR2 and CCR5 detected on ascitic monocytes, in comparison to that of normal donors, migration response to their ligand RANTES was impaired [
16].
Diabetes mellitus type 2 is often associated with obesity. It has been reported that the Hh signaling pathway was less active in obese compared to lean mice. Two models have been tested, mice invalidated in leptine (ob/ob) and high-fat diet induced obesity. So far all experiments leading to a stimulation of Hh signaling in vivo result in an enlarged body mass and conversely, an inhibition of the pathway prevents weight gain [
11]. We did not observe significant differences between CAD−DM and CAD+DM with regards to BMI. Therefore, it is unlikely that an excess of adipose mass, which characterizes obesity and is often associated with diabetes, may play a role in disregulation of Hh pathway.
Moreover, diabetes mellitus is also accompanied by a systemic low-grade inflammation and it is an important factor in accelerating atherosclerosis. It has been reported that LPS-induced Ptc expression in macrophages after 18 h in culture [
41]. Thus, low-grade inflammation could upregulate expression of Ptc on circulating monocytes of diabetes patients. In addition, it has been previously shown in
Drosophila that to achieve maximal pathway activity, large excess (more than 50-fold) of Smo over Ptc is required [
36]. If similar regulation of Hh signaling applies to the human system, the strong upregulation of Ptc and preserved expression level of Smo in diabetic patients could explain the observed defect in Hh signaling.
Monocytes/macrophages are recognized as one of the principal cell types in the pathology of atherosclerosis [
43,
44]. Accumulating evidence suggests that the Hh pathway is involved in peripheral immunity and tissue remodeling [
21,
23]. Expression of Shh and Ptc is upregulated in small bowel allografts undergoing chronic rejection. Systemic treatment with a neutralizing anti-Shh antibody reduced tissue remodeling, fibrosis and vascular occlusion of the intestinal grafts [
7]. The cells that expressed Ptc in the intestinal grafts undergoing chronic rejection were mainly macrophages. In a recent study using an ApoE
−/−-based mouse model of atherosclerosis, inhibition of Shh using specific antibodies resulted in a pro-atherosclerotic phenotype secondary to enhanced lipid uptake in macrophages [
3]. Our novel data do support the idea that inhibition of Shh may be pro-atherogenic. Macrophages in human atherosclerotic plaques express high levels of receptor Ptc. The diabetes-related inhibition of macrophages may prevent their clearance from atherosclerotic plaques. This could be a novel molecular mechanism promoting atherosclerosis; however, its functional relevance and extent in the pathogenesis of human atherosclerosis remains to be determined.
Thus, our data represent the first example of an endogenous metabolic abnormality, namely DM, which is associated with a functional inhibition of the Hh signaling pathway. Likewise, this impaired monocyte function is likely to be associated with impaired arteriogenesis and impaired wound healing [
35,
39]. Identification of novel signal transduction pathways regulating monocyte chemotaxis can indicate unique targets for preventive therapies for treatment of chronic inflammatory diseases.