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Increased plasma levels of the VEGF165b splice variant are associated with the severity of nailfold capillary loss in systemic sclerosis
  1. Mirko Manetti1,2,
  2. Serena Guiducci2,
  3. Eloisa Romano2,
  4. Silvia Bellando-Randone2,
  5. Gemma Lepri2,
  6. Cosimo Bruni2,
  7. Maria Letizia Conforti2,
  8. Lidia Ibba-Manneschi1,
  9. Marco Matucci-Cerinic2
  1. 1Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Florence, Italy
  2. 2Department of Experimental and Clinical Medicine, Section of Internal Medicine and Division of Rheumatology, Azienda Ospedaliero-Universitaria Careggi, University of Florence, Florence, Italy
  1. Correspondence to Dr Mirko Manetti, Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Largo Brambilla 3, Florence I-50134, Italy; mirkomanetti{at} MM and SG contributed equally to this work.

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In systemic sclerosis (SSc), Raynaud's phenomenon is the earliest clinical manifestation paralleled by nailfold capillaroscopic alterations that may occur months or even years before the onset of fibrosis.1 This evidence suggests a crucial role of microangiopathy characterised by a progressive loss of capillaries with the formation of avascular areas.1 ,2 Consequent chronic tissue hypoxia leads to skin ulcers and gangrene that heavily burden patients’ quality of life.1 ,2 Vascular endothelial growth factor-A (VEGF-A) is overexpressed in SSc skin, and increased circulating levels of VEGF-A correlate with the severity of nailfold capillary loss.2–4 It has been demonstrated that the VEGF-A primary transcript can be alternatively spliced in its terminal exon, producing two distinct mRNA splice variants that are translated to the proangiogenic VEGF165 and antiangiogenic VEGF165b isoforms.5 These two isoforms bind to the tyrosine kinase receptor VEGFR-2 with the same affinity, but binding of VEGF165b results in an insufficient tyrosine phosphorylation/activation of VEGFR-2 and incomplete or transient downstream signalling, which lead to an impaired angiogenic response.5 ,6 Recently, we have provided the first evidence that dermal expression and plasma levels of VEGF165b are raised in SSc patients, and VEGF165b overexpression prevents SSc dermal microvascular endothelial cells from entering a suitable angiogenic programme in vitro.6 ,7 However, whether VEGF165b levels may reflect the extent of peripheral vascular damage in SSc remains to be determined. In the present study, we evaluated the circulating levels of VEGF165b in SSc and their possible correlation with microvascular involvement.

Sixty-nine SSc patients entered the study. Patients were classified as limited cutaneous SSc (lcSSc; n=41) or diffuse cutaneous SSc (dcSSc; n=28)8 and assessed as previously described.9 Nailfold videocapillaroscopy (NVC) was performed on all 10 fingers by a single rheumatologist (SG), and images were scored by two blinded experienced examiners (GL and CB) who divided patients into three capillaroscopic patterns (ie, early, active and late).9 ,10 In table 1, patients’ characteristics are summarised. Forty-one age-matched and sex-matched healthy subjects served as controls. Plasma VEGF165b levels were measured by quantitative colorimetric sandwich ELISA using reagents from R&D Systems (Minneapolis, Minnesota, USA). The non-parametric Mann–Whitney U-test for non-related samples was used for statistical analysis. For all comparisons between SSc subsets, Bonferroni adjustment was used for multiple comparisons. Values of p after this adjustment for multiple testing are termed as ‘padj’. A p value of less than 0.05 was considered statistically significant.

Table 1

Demographical and clinical characteristics of SSc patients

In agreement with our previous report,7 plasma levels of VEGF165b were significantly increased in SSc patients compared with controls (p=0.004; figure 1A). VEGF165b levels in lcSSc and dcSSc were significantly higher than in controls (p=0.015 and p=0.011, respectively), but not different between the two subsets (figure 1A). VEGF165b plasma levels were significantly higher in SSc patients with ‘late’ NVC pattern than in those with ‘early’ and ‘active’ patterns (both padj=0.02; figure 1B). VEGF165b was increased in patients with ‘active’ NVC pattern compared with those with ‘early’ pattern, but this difference did not reach statistical significance (figure 1B). Moreover, VEGF165b levels were significantly raised in SSc patients either with ‘active’ or ‘late’ NVC pattern than in controls (p=0.04 and p<0.001, respectively), whereas no difference was found between SSc patients with ‘early’ NVC pattern and controls (figure 1B). As the features of each NVC pattern are different, but somewhat overlapping between ‘early’, ‘active’ and ‘late’ groups,10 plasma levels of VEGF165b were also correlated with the single NVC changes. Increased plasma levels of VEGF165b correlated significantly with the absence of microhaemorrhages (padj=0.001) and the presence of ramified/bushy capillaries (padj=0.001) and avascular areas (padj=0.02) (figure 1C). No significant association was found with other clinical and laboratory parameters.

Figure 1

(A,B) Plasma levels of VEGF165b in healthy controls, patients with systemic sclerosis (SSc), limited cutaneous SSc (lcSSc), diffuse cutaneous SSc (dcSSc) and according to ‘early’, ‘active’ and ‘late’ nailfold videocapillaroscopy patterns. (C) Plasma levels of VEGF165b in SSc patients in accordance with the presence or absence of single capillaroscopic changes (ie, microhaemorrhages, giant capillaries, ramified or bushy capillaries and avascular areas). VEGF165b levels in plasma samples were determined by quantitative colorimetric sandwich ELISA. Each sample was measured in duplicate. Data are shown as box plots. Each box represents the 25–75th percentiles. Lines outside the boxes represent the 10–90th percentiles. White lines inside the boxes represent the median, circles represent the outliers and asterisks represent the extreme values. The non-parametric Mann–Whitney U-test for non-related samples was used to analyse the differences in VEGF165b levels between groups. For all comparisons between SSc subsets, Bonferroni adjustment was used for multiple comparisons. NS, not significant; padj, adjusted p value; +, present; −, absent.

Our findings demonstrate for the first time that in SSc, increased plasma levels of the antiangiogenic VEGF165b isoform are associated with the severity of capillary architectural derangement and loss. However, as the NVC phenotype can change during the disease course, prospective follow-up investigations will be required. Together with previous data,6 ,7 these results suggest that in SSc, the VEGF165b splice variant might actively participate in the loss of microvessels. In SSc, the regulation of VEGF-A pre-mRNA splicing might become a new target for the control of the progression of peripheral microvasculopathy.


The authors thank Claudia Ceccarelli for her excellent technical assistance in setting up the ELISA on plasma samples.



  • Contributors All authors were involved in drafting the article or revising it critically for important intellectual content and all authors approved the final version to be published. Study conception and design: MM, SG, ER, LI-M and MM-C. Acquisition of data: MM, SG, ER, SB-R, GL, CB, MLC and MM-C. Analysis and interpretation of data: MM, SG, ER, SB-R, GL, CB, LI-M and MM-C.

  • Funding This study has been supported by grants from the University of Florence (Progetti di Ricerca di Ateneo to MM-C).

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval The study was approved by the institutional review board. All subjects gave written informed consent.

  • Provenance and peer review Not commissioned; externally peer reviewed.