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Association between a stromal cell-derived factor 1 (SDF-1/CXCL12) gene polymorphism and microvascular disease in systemic sclerosis
  1. M Manetti1,2,
  2. V Liakouli3,
  3. C Fatini4,
  4. P Cipriani3,
  5. C Bonino5,
  6. S Vettori6,
  7. S Guiducci2,
  8. C Montecucco5,
  9. R Abbate4,
  10. G Valentini6,
  11. M Matucci-Cerinic2,
  12. R Giacomelli3,
  13. L Ibba-Manneschi1
  1. 1
    Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy
  2. 2
    Department of Biomedicine, Division of Rheumatology, AOUC and Excellence Centre for Research, Transfer and High Education DENOthe, University of Florence, Florence, Italy
  3. 3
    Department of Internal Medicine and Public Health, Division of Rheumatology, University of L’Aquila, L’Aquila, Italy
  4. 4
    Department of Medical and Surgical Critical Care, Thrombosis Centre, University of Florence, Florence, Italy
  5. 5
    Division of Rheumatology, University of Pavia, IRCCS Policlinico S Matteo, Pavia, Italy
  6. 6
    Division of Rheumatology, II University of Naples, Naples, Italy
  1. Professor L Ibba-Manneschi, Department of Anatomy, Histology and Forensic Medicine, University of Florence, Viale G B Morgagni 85, 50134 Florence, Italy; ibba{at}unifi.it

Abstract

Objective: To investigate the possible implication of SDF1-3′ polymorphism in systemic sclerosis (SSc) susceptibility or clinical phenotype, or both.

Methods: 150 patients with SSc and 150 controls were enrolled. Skin involvement, autoantibodies, interstitial lung disease, pulmonary arterial hypertension (PAH), scleroderma renal crisis, past and/or current skin ulcers were assessed. Genotyping was performed by PCR-RFLP.

Results: Genotype distribution and allele frequency were similar in SSc and controls. SDF1-3′A allele and SDF1-3′GA/AA genotype frequencies were significantly higher in SSc-PAH than in SSc-non-PAH (33.3% vs 18.3%, p = 0.01) and in SSc with skin ulcers than in SSc without ulcers (27.3% vs 16.9%, p = 0.03). The SDF1-3′A allele influenced the predisposition to SSc-related PAH (OR = 2.52, 95% CI 1.11 to 5.69, p = 0.02) and skin ulcers (OR = 2.31, 95% CI 1.18 to 4.52, p = 0.01). After adjustment for age and gender, the SDF1-3′A allele remained a susceptibility factor for the SSc-related vascular manifestations (PAH: OR = 2.37, 95% CI 1.04 to 5.42, p = 0.04; ulcers: OR = 2.33, 95% CI 1.78 to 4.62, p = 0.01).

Conclusion: The SDF1-3′A allele is significantly associated with microvascular involvement in SSc.

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Systemic sclerosis (SSc) is a connective tissue disorder characterised by early generalised microangiopathy which evolves into fibrosis of the skin and internal organs.1 2 Vascular complications, including ischaemic ulcers, pulmonary arterial hypertension (PAH) and scleroderma renal crisis (SRC), have emerged as leading causes of disability and mortality in SSc.3

Endothelial cell (EC) injury and apoptosis occur at an early disease stage, leading to an irregular chaotic architecture and reduced density of the capillary network.1 2 Vascular changes result in decreased capillary blood flow causing tissue hypoxia, which may manifest clinically as digital ulcers and gangrene.3 In SSc, although tissue hypoxia is a major stimulus to initiate the formation of new capillary vessels from pre-existing vessels (angiogenesis), there is no evidence of significant angiogenesis.2 Furthermore, vasculogenesis is also impaired in SSc, with altered numbers and functional defects of endothelial progenitor cells (EPCs).4

The CXC chemokine stromal cell-derived factor 1 (SDF-1/CXCL12) and its receptor CXCR4 regulate specific steps in new vessel formation.5 Experimental deficiency in the SDF-1 or CXCR4 gene in the embryo results in a lethal phenotype characterised by defective development of the cardiovascular system.6 SDF-1/CXCR4 binding promotes EC proliferation, as well as EC resistance to apoptosis, cytoskeletal reorganisation, migration and formation of a vascular lumen.5 6 Moreover, the SDF-1/CXCR4 axis plays a major role in CD34+ EPC mobilisation into peripheral blood and homing to ischaemic tissues.7

In SSc, a perturbation of the SDF-1/CXCR4 system was recently implicated in the pathogenesis of microvascular abnormalities and defective vascular repair.8 9

The SDF-1 gene, which is located on chromosome 10q11, is polymorphic, with a G-to-A transition at position 801 in the 3′-untranslated region of cDNA encoding SDF-1β, one of the two isoforms of SDF-1, referred to below as SDF1-3′ polymorphism (rs1801157).10 Interestingly, this genetic variant seems to regulate SDF-1 gene transcriptional activity and has been associated with the levels of CD34+ progenitor cells mobilised into peripheral blood in humans.11 12

The purpose of our work was to investigate the possible implication of SDF1-3′ polymorphism in SSc susceptibility and/or clinical phenotype in a multicentre cohort of Italian Caucasian patients with SSc.

PATIENTS AND METHODS

Patients and controls

One hundred and fifty patients with SSc attending the outpatient clinic of the divisions of rheumatology of the Universities of L’Aquila, Florence, Pavia and II University of Naples were enrolled in the study. Overlap syndromes were excluded. One hundred and fifty healthy subjects, comparable with patients for age and sex and residing in the same geographical areas, were recruited from blood donors. Exclusion criteria for controls were history of autoimmune or any other systemic disease, or both. All subjects were Italian Caucasian, unrelated to each other and gave written informed consent to participate in the study as approved by the local ethics committees.

Clinical assessment

An extensive clinical profile was established for each patient by reviewing their medical records. Limited or diffuse cutaneous SSc (lcSSc/dcSSc) subsets were defined according to LeRoy et al.13 Past and/or current fingertip ulcers and other skin ulcers (eg, at the lower extremities, elbows, or forearms) were recorded. Patients with other comorbidities that can cause skin ulceration (eg, diabetes mellitus, chronic obstructive arteriopathy) were excluded. Disease duration was calculated since the first non-Raynaud’s symptom. Antinuclear, anticentromere and anti-topoisomerase I antibodies were determined following standard techniques. Organ involvement was assessed according to international guidelines.14 Interstitial lung disease (ILD) was studied with pulmonary function tests and high-resolution computed tomography and was diagnosed when forced vital capacity and carbon monoxide transfer factor (Tlco) were ⩽70%. In the presence of Tlco reduction alone, patients were investigated for PAH. A systolic pulmonary artery pressure >40 mm Hg by echocardiography was considered to be pathological. Right heart catheterisation was performed to confirm PAH, defined as a mean pulmonary artery pressure >25 mm Hg at rest or >30 mm Hg during exercise with normal pulmonary capillary wedge pressure (<15 mm Hg).15 SRC was defined according to the presence of new-onset accelerated hypertension with evidence of renal function impairment, microangiopathic haemolysis or significant end-organ damage.

Genotyping

DNA was extracted from peripheral blood leucocytes according to standard procedures. SDF1-3′ polymorphism was genotyped with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, using forward primer 5′-CAGTCAACCTGGGCAAAGCC-3′ and reverse primer 5′-AGCTTTGGTCCTGAGAGTCC-3′ (GenBank accession no NM_000609). DNA (60 ng) was amplified using AccuPrime SuperMix II (Invitrogen, Carlsbad, California, USA). PCR conditions were: initial denaturation at 94°C for 2 min, 35 cycles of 94°C for 30 s, 58°C for 30 s and 68°C for 1 min, with a final extension at 68°C for 5 min using a TC-512 thermocycler (Techne, Cambridge, UK). PCR products were digested with MspI restriction endonuclease (M-Medical, Milan, Italy) and separated by agarose gel electrophoresis (fig 1).

Figure 1 Analysis of SDF1-3′ gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Genomic DNA was amplified using specific primer sequences which generate a 302 bp fragment containing the polymorphic site. PCR products were digested with the restriction endonuclease MspI and subsequently separated by 1% agarose gel electrophoresis with ethidium bromide staining and visualised under ultraviolet transillumination. The SDF1-3′G wild-type allele resulted in two fragments of 202 and 100 bp, whereas the SDF1-3′A gene variant resulted in one fragment of 302 bp because of the elimination of the MspI restriction site. Results of a representative experiment are shown. Samples 1 and 2 have the heterozygous GA genotype, sample 3 the AA genotype and samples 4 and 5 the GG genotype.

Statistical analysis

Statistical analysis was performed using SPSS software, version 11.5. Genotype distribution and allele frequencies in both patients and controls were compared using χ2 analysis. A χ2 test was used to compare the observed numbers of each genotype with those expected for a population in Hardy–Weinberg equilibrium. The association between SDF1-3′ polymorphism and SSc or clinical phenotypes was assessed using univariate analysis under a dominant genetic model of inheritance, which compares subjects with one or more polymorphic alleles with a baseline group with no polymorphic alleles (ie, SDF1-3′GA/AA vs SDF1-3′GG). Variables such as age and gender were included into the multivariate model in order to evaluate their confounding effect. The odds ratio (OR) with 95% confidence interval (95% CI) was determined. p Values <0.05 were considered significant.

RESULTS

Table 1 gives the demographic and clinical characteristics of the study group.

Table 1 Demographic, clinical and serological characteristics of patients with SSc and control subjects

SDF1-3′ polymorphism genotype distribution and allele frequency in patients with SSc and controls

We found no deviation from the Hardy–Weinberg equilibrium both in patients with SSc and controls. As the percentage of homozygotes for SDF1-3′A allele was low, subjects carrying the SDF1-3′AA genotype were combined with those carrying the SDF1-3′GA genotype for statistical analysis.

The SDF1-3′A allele frequency found in controls (24.0%) was in the range of that previously reported in the European Caucasian populations.10 No significant difference in SDF1-3′A rare allele frequency between patients with SSc and controls was detected (21.3% vs 24.0%). Similarly, no difference in the percentage of subjects carrying the SDF1-3′A allele between patients and controls was found (38.7% vs 43.3%) (table 2). Higher, even if not significant, SDF1-3′A allele frequency and SDF1-3′GA/AA genotype percentage were seen in dcSSc than in lcSSc and in anti-topoisomerase I-positive compared with anti-topoisomerase I-negative SSc (table 2). No difference in genotype distribution and SDF1-3′A allele frequency was found between SSc-ILD and SSc-non-ILD (table 2).

Table 2 Genotype distribution of the SDF1-3′ gene polymorphism and frequency of the SDF1-3′A allele in control subjects, patients with SSc and SSc clinical subsets

SDF1-3′ polymorphism and SSc vascular phenotype

We observed a significant difference in SDF1-3′A allele frequency between SSc-PAH and SSc-non-PAH (33.3% vs 18.3%, p = 0.01), as well as between patients with SSc with skin ulcers and those who never experienced ulcers (27.3% vs 16.9%, p = 0.03) (table 2). Furthermore, the percentage of subjects carrying the SDF1-3′A allele was significantly higher in SSc-PAH than in SSc-non-PAH (56.7% vs 34.2%, p = 0.02) and in patients with skin ulcers than in those without history of ulcers (50.0% vs 30.2%, p = 0.01). The frequency of the SDF1-3′A allele was higher, even if not significant, in SSc-SRC compared with SSc-non-SRC (29.2% vs 20.7%) (table 2).

The SDF1-3′A allele influenced the predisposition to both SSc-related PAH (OR = 2.52, 95% CI 1.11 to 5.69, p = 0.02) and skin ulcer development (OR = 2.31, 95% CI 1.18 to 4.52, p = 0.01). After adjustment for age and gender, the presence of the SDF1-3′A allele remained a susceptibility factor for both SSc-related vascular manifestations (PAH: OR = 2.37, 95% CI 1.04 to 5.42, p = 0.04; skin ulcers: OR = 2.33, 95% CI 1.78 to 4.62, p = 0.01). Interestingly, when we considered subjects with the simultaneous presence of PAH and skin ulcers, 15/19 patients (79.0%) carried the SDF1-3′A rare allele in comparison with 4/19 (21.0%) who were homozygotes for the SDF1-3′G wild-type allele.

DISCUSSION

This study demonstrates an association between an SDF-1 gene single nucleotide polymorphism (SNP) and the vascular phenotype of SSc.

Increasing evidence suggests that SSc is a complex polygenic disorder resulting from multiple interactions between genetic and environmental factors. Candidate genes for SSc include genes encoding proteins involved in fibrosis, vasculopathy and autoimmunity, fully reflecting disease heterogeneity and complexity.16 In recent years several studies have shown that SNPs may not have a causative role in SSc pathogenesis, but rather may be relevant in disease progression or clinical phenotype, or both.16 In particular, polymorphisms of genes regulating EC plasticity/function have previously been shown to be associated with SSc-PAH.16

SDF-1 and its receptor CXCR4 play a major role in the normal maintenance and remodelling of the vascular system.5 6 After induction of peripheral ischaemia, SDF-1 expression is upregulated in hypoxic tissues, stimulating ECs towards an angiogenic response. Furthermore, an SDF-1 gradient is established, thus facilitating both EPC mobilisation into peripheral blood and their homing to ischaemic tissues and incorporation into new vessels.7

We recently demonstrated an altered expression of both SDF-1 and CXCR4 in skin and ECs from patients with SSc.8 In addition, we showed an implication of SDF-1/CXCR4 axis in the impaired commitment of SSc bone marrow-derived mesenchymal stem cells towards EC lineage.9

This study investigated whether a common SDF-1 gene variant may be implicated in SSc susceptibility or disease phenotypes, or both. Indeed, recent studies indicated that SDF1-3′ polymorphism may be associated with SDF-1 gene transcriptional activity and CD34+ progenitor cell mobilisation into peripheral blood.1012 In our case–control cohort, the frequency of SDF1-3′ genotypes and alleles was similar in SSc and controls. Interestingly, within our SSc population the presence of the SDF1-3′A rare allele was significantly over-represented in patients with vascular complications (PAH, ischaemic ulcers). Moreover, although the small number of patients with SRC might have precluded the detection of a significant association, the SDF1-3′A allele was found with a higher frequency in SSc-SRC.

However, several factors should be taken into consideration when interpreting these results. First, disease duration may account for the occurrence of vascular manifestations. Thus, a prospective follow-up is continuing. Second, because of the relatively low prevalence of PAH, the association will need to be confirmed in larger SSc-PAH populations. Finally, we cannot exclude the possibility that a linkage disequilibrium between SDF1-3′ polymorphism and other SNPs spanning the SDF-1 gene might affect SSc susceptibility and/or vascular phenotype. Further investigation is needed to deal with this important topic. Moreover, the potential functional role of SDF1-3′ polymorphism in influencing the defective vascular repair capability of ECs/EPCs remains of major interest in SSc.

In conclusion, our findings show that SDF1-3′ polymorphism may modulate SSc vascular phenotype, further arguing for a critical role of the SDF-1/CXCR4 axis in the vascular component of SSc pathogenesis. Further studies will be required to confirm the association reported here in independent cohorts of patients with SSc.

REFERENCES

Footnotes

  • Competing interests: None.

  • Funding: This study has been supported by grants from the Ministero Italiano dell’Università e della Ricerca (MIUR) and the Associazione per lo studio della Sclerosi Sistemica e delle Malattie Fibrosanti (ASSMaF onlus).

  • Ethics approval: Approved by the local ethics committees.

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