Barbara Ruaro

Laser speckle contrast analysis: a new method to evaluate peripheral blood perfusion in systemic sclerosis patients

Objective The aim of this pilot study was to assess peripheral blood perfusion (PBP) by a new technique, the laser speckle contrast analysis (LASCA), in systemic sclerosis (SSc) patients showing different patterns of nailfold microangiopathy. Correlations between LASCA and single laser Doppler flowmetry (LDF) analysis were also checked. Methods Sixty-one SSc patients and 61 healthy subjects were enrolled. PBP was evaluated using LASCA and LDF. Scleroderma patterns and microangiopathy evolution score (MES) were assessed by nailfold videocapillaroscopy (NVC). Results As detected by LASCA and LDF, PBP was lower in SSc patients than in healthy subjects (p<0.0001), showing SSc patients with the ‘Early’, ‘Active’ or ‘Late’ NVC pattern a progressively lower PBP (p=0.04 and p=0.002, respectively). There was a negative correlation between PBP and MES values (p=0.006 and p=0.002 for LASCA and LDF, respectively). A positive correlation was detected between LASCA and LDF values, in all subjects (p<0.0001). However, LASCA evaluates larger skin areas, is significantly less time consuming, is more accepted by patients and shows lower intra-operator variability than LDF. Conclusions LASCA detected lower PBP in SSc patients than in healthy subjects, and for the first time, LASCA perfusion values were found correlated with progression of NVC patterns of microangiopathy.

Correlations between nailfold microangiopathy severity, finger dermal thickness and fingertip blood perfusion in systemic sclerosis patients

Objective The aim of this study was to identify possible correlations between nailfold microangiopathy severity, finger dermal thickness (DT) and fingertip blood perfusion (FBP) in systemic sclerosis (SSc) patients. Methods Fifty-seven SSc patients and 37 healthy subjects were enrolled. All patients were evaluated by nailfold videocapillaroscopy (NVC) to classify and score the severity of microangiopathy. Both modified Rodnan skin score (mRss) and skin high-frequency ultrasound were used to detect finger DT. Laser Doppler flowmetry (LDF) was employed to detect FBP. Results A positive correlation was found between nailfold microvascular damage severity and both ultrasound-DT (p=0.028) and mRss values (p<0.0001). In particular, both ultrasound-DT and mRss were found progressively higher in patients with ‘Early’, ‘Active’ or ‘Late’ NVC pattern of microangiopathy. A negative correlation was observed between nailfold microvascular damage severity and FBP (p<0.0001), showing the lowest FBP of the patients with more advanced NVC patterns. A negative correlation was observed between FBP, and both ultrasound-DT (p=0.007) and mRss values (p=0.0002). SSc patients showed a higher ultrasound-DT at the level of the fingers, as well as a lower FBP than healthy subjects (p<0.0001). Conclusions This study demonstrates a relationship between nailfold microangiopathy severity, DT and FBP in SSc patients.

Longterm treatment with endothelin receptor antagonist bosentan and iloprost improves fingertip blood perfusion in systemic sclerosis.

Objective. To evaluate the longterm effects of endothelin-1 (ET-1) antagonism on peripheral blood perfusion (PBP) in patients with systemic sclerosis (SSc). Methods. Twenty-six patients with SSc already receiving cyclic intravenous iloprost (ILO) for severe Raynaud phenomenon were enrolled. Thirteen patients continued the treatment for a further 3 years (ILO group) and 13 patients, because of the appearance of digital ulcers, received in addition bosentan (BOS; 125 mg twice/day) for 3 years (ILO + BOS group). Both PBP at fingertips and nailfold microangiopathy were evaluated yearly by laser Doppler flowmetry and nailfold videocapillaroscopy, respectively. Results. A progressive significant increase of PBP was observed in the ILO + BOS group during the 3 followup years (p = 0.0007, p = 0.0002, p = 0.01, respectively). In contrast, an insignificant progressive decrease of PBP was observed in the ILO group. Difference of perfusion between the PBP evaluations at basal temperature and at 36°C (to test capillary dilation capacity), was found progressively decreased during the 3-year followup only in the ILO group (p = 0.05, p = 0.26, p = 0.09, respectively). A progressive increase of nailfold capillary number was observed only in the ILO + BOS group after 2 and 3 years of followup (p = 0.05). Conclusion. Longterm treatment of SSc patients with ET-1 antagonism, in combination with ILO, seems to increase fingertip blood perfusion, as well as both capillary dilation capacity and number.

Short-term follow-up of digital ulcers by laser speckle contrast analysis in systemic sclerosis patients

OBJECTIVE To monitor in systemic sclerosis (SSc) the evolution of digital ulcer (DU) status by analysing blood perfusion (BP) using laser speckle contrast analysis (LASCA). METHODS Hand BP was recorded by LASCA in twenty SSc patients with recent onset fingertip DUs before and after 10days of local/systemic treatment. Regions of interest (ROIs) to analyse BP were created al the level of ulcer, peri-ulcer, periungual and fingertip areas. Visual analogue pain scale (VAS) was also administered to patients before and after follow-up. RESULTS A statistically significant increase of BP was observed from T0 to T1 in the ROIs created at the level of the ulcer area (p<0.0001), as well as a significant decrease of BP was observed in the peri-ulcer area (p<0.0001). A statistically significant decrease of both ulcer size (p<0.0001) and VAS (p=0.001) was observed, whereas no significant variation of both periungual and fingertip BP was detected. CONCLUSIONS LASCA may safely monitor DU evolution in SSc patients, by evaluating its blood perfusion and area during standard treatment. This may be useful to monitor DU evolution during treatment in clinical trials.

Effects of Longterm Treatment with Bosentan and Iloprost on Nailfold Absolute Capillary Number, Fingertip Blood Perfusion, and Clinical Status in Systemic Sclerosis

Objective. To quantify in patients with systemic sclerosis (SSc) the absolute nailfold capillary number/mm (the absolute number of capillaries, observable in the first row, in 1 mm per field) and fingertip blood perfusion (FBP) during longterm therapy with the endothelin receptor antagonist bosentan (BOSE) and the synthetic analog of prostacyclin PGI2 iloprost (ILO) by multiple diagnostic tools. Observed values were correlated with clinical outcomes. Methods. Thirty patients with SSc already receiving intravenous ILO (80 μg/day) for 5 continuous days (every 3 mos) were recruited in the clinic. Fifteen patients continued such treatment (ILO group), while in 15 patients BOSE (125 mg twice/day) was added (ILO + BOSE group) because of the onset of pulmonary arterial hypertension or digital ulcers (DU). The followup period was 4 years (T0–T4). Every year the following were evaluated: absolute nailfold capillary number/mm by nailfold videocapillaroscopy, FBP by laser Doppler flowmetry, DU incidence, DLCO, systolic pulmonary arterial pressure (sPAP), renal arterial resistive index, and other biomarkers. From T2 to T4, laser speckled contrast analysis was added. Nonparametric tests were used for statistical analysis. Results. Limited to the ILO + BOSE group, absolute capillary number/mm and FBP showed a progressive increase independently from other variables. In addition, during followup there was a significant reduction (80%) in the incidence of new DU, whereas DLCO and sPAP did not worsen. Conclusion. The study shows in patients with SSc with up to 4 years of combined therapy a progressive significant recovery in structure and function of microvasculature linked to improved clinical outcomes, independent of disease severity.

Progression of nailfold microvascular damage and antinuclear antibody pattern in systemic sclerosis.

Objective. This study evaluates possible correlations between the pattern of antinuclear antibodies (ANA) on indirect immunofluorescence (IIF) testing and nailfold microangiopathy stage (early, active, and late stage) in systemic sclerosis (SSc). Patients with SSc were followed prospectively to monitor progression of microvascular damage. Methods. The ANA pattern on IIF was searched in 42 patients with SSc showing an early pattern of nailfold microangiopathy at baseline, and was followed using nailfold videocapillaroscopy (NVC) for a median time of 91 months. Results. Among patients whose microangiopathy showed a rapid progression from early to late pattern on NVC, the IIF pattern was fine-speckled + nucleolar (Scl-70+) in 44%, centromeric in 33%, nucleolar in 11%, and homogeneous in 11% of patients with SSc. Antitopoisomerase I antibodies were significantly more frequent (57%) in patients with late pattern of microangiopathy on NVC. The median time of progression from early to active disease was significantly lower in patients with both fine-speckled + nucleolar and nucleolar ANA positivity. The severity of microangiopathy was higher in patients with the nucleolar pattern on IIF. Patients already showing a slight reduction of capillary number at baseline were likely to have either the nucleolar or the fine-speckled + nucleolar pattern on IIF. Of note, 37% of patients still showing the early microangiopathy pattern on NVC at the end of the followup were ANA-negative. Conclusion. ANA-negative patients with SSc display a slower progression of nailfold microangiopathy characterized by the early pattern on NVC. Progression to the late NVC pattern (more advanced stage of microvascular damage) seems to be associated with a different autoantibody pattern on IIF (fine-speckled + nucleolar pattern being the most prevalent).

Nailfold Capillaroscopy and Clinical Applications in Systemic Sclerosis.

Capillary microscopy is a safe and non‐invasive tool to evaluate the morphology of the microcirculation typically affected in SSc. Next to being paramount for the “(very) early” diagnosis of SSc eyes are also geared toward capillaroscopy with the aim to be able to use it as a biomarker, especially in the prediction of future occurrence of DU. The following review will explain what capillary microscopy is and will focus additionally on studies evaluating the association between capillaroscopy and DU.

Quantitative alterations of capillary diameter have a predictive value for development of the capillaroscopic systemic sclerosis pattern

Objective. To quantify earlier capillary diameter abnormalities, observed by nailfold videocapillaroscopy (NVC), in primary Raynaud phenomenon (PRP) subjects compared with RP subjects later evolved to systemic sclerosis (SSc)-associated secondary Raynaud phenomenon (SRP). Methods. There were 6112 NVC images of 191 subjects analyzed at baseline and after a mean followup of 42.77 ± 35.80 months. We selected 48 patients affected by SRP and 143 matched controls confirmed with PRP. The diameter of the most dilated limbs (arterial, venous, and apical) was measured in 16 images per subject. Statistical analysis was performed using nonparametric tests. The threshold values for capillary diameters associated with the development of SSc-associated SRP were determined through receiver-operating characteristic curves. Results. Mean capillary diameter values were significantly different for arterial, venous, and average diameters (mean value of arterial, venous, and apical) between patients with PRP and SRP (p < 0.0001). These alterations were found to be independent predictors for disease development (p = 0.015). Threshold values of 30 µm (area under the curve = 0.802, sensitivity/specificity = 0.85/0.63) to 31 µm were identified for average, arterial, and venous diameters, with a shortening effect on time to disease development. Conclusion. The study showed that capillary diameter is an independent predictor for development of SSc-associated SRP. Progression to SRP is unlikely for subjects affected by RP when average capillary diameter is under 30 μm. Subsequently, the execution of the qualitative/quantitative integrated analysis should be part of the NVC followup of RP subjects.

Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

Background Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Methods Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. Results ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were contrasted by ETA/BRA treatment in both cultured cell types. Conclusion ET-1 seems to induce the M2 phenotype in cultured human macrophages, a process apparently contrasted by the action of the ETA/BRA, suggesting possible clinical implications in those fibrotic diseases characterized by increased ET-1 concentrations, such as systemic sclerosis but also type 2 diabetes.

Macrocirculation versus microcirculation and digital ulcers in systemic sclerosis patients

In their research published in this issue, Lüders et al. aimed to compare, in SSc patients, the presence of hand-altered arteries with nailfold capillaroscopy (NC) and with clinical signs of digital ischaemia, that is, digital ulcers (DUs) or pitting scars (PSs). The authors confirmed the already reported presence of structural changes in the arteries at the level of the wrists and hands, as detected by colour Doppler ultrasonography (CDUS) [1, 2]. These changes are highly frequent in SSc patients but absent in healthy subjects or patients with primary RP [3]. Furthermore, ulnar artery occlusions have already been identified as a risk factor for new DUs [2]. Lüders et al. [1] analysed 79 SSc patients and showed that almost 40% of all assessable arteries, almost 50% of all proper palmar digital arteries (PPDAs), but only 15% of proximal arteries showed narrowed or occluded lumens [1]. More specifically, fingerwise analyses presented significant coincidence of pathological CDUS findings and DUs/PSs (Fig. 1B). Interestingly, patients with a nonSSc-specific or early NC pattern tended to have a nonsignificant lower number of pathological arteries in CDUS than patients with active or late NC patterns. However and unexpectedly, no significant difference was observed in the number of altererd arteries (macrocirculation) in patients with the late NC pattern (microcirculation), compared with the other patterns (non-SSc, early and active patterns) put together. In addition and unexpectedly, there was no link between reduced capillary density (<7/ mm at NC), present in 79% of assessable fingers, and the existence of pathological vessels in CDUS. Therefore, the dissociation observed in the present study seems partially in contrast with a very recent multicentre study, which analysed 468 SSc patients, and defined the mean number of capillaries per millimetre, together with the number of DUs, as well as signs of severe digital ischaemia at baseline, as significant risk factors for the development of new DUs during a 6-month follow-up [4]. These results confirmed a previous study that offered a simple prognostic index for digital trophic lesions for daily use in SSc clinics, limited to the mean score of capillary loss [5]. At this point, one should expect that the microvascular counterpart, namely the presence of the late NC pattern indicating the most advanced nailfold capillary loss in SSc, should be coincident since almost 50% of all the PPDAs showed narrowed or occluded lumens, as a consequence of the advanced peripheral macrovessel intimal fibrosis [6]. However, assuming a link between macrovasculopathy and microvasculopathy in SSc, the problems arising from the controversies related to the morphological vascular aspects might be better clarified if integrated by functional analysis, through quantification of the blood flow changes in both peripheral arteries and capillaries. Rosato et al. [7] analysed SSc patients and healthy matched controls, in order to assess both morphology and blood flow of PPDAs by CDUS, skin blood perfusion, digital artery pulsatility and the relationships with NC patterns. Interestingly, CDUS was found to be pathological in 69% of SSc patients and in none of the healthy controls. SSc patients with low microvascular damage (the early NC pattern) showed a normal morphology of PPDAs, but the blood flow was found to be reduced and vascular resistance was found to be increased. At this stage, the mean perfusion evaluated by laser Doppler perfusion imaging and the digital artery pulsatility was found to be reduced. Finally, the US hand changes appeared associated with microvascular damage progression (active and late NC patterns), while, in contrast, the PPDA blood flow progressively decreased. Interestingly, we described the simultaneous presence of macrovascular and microvascular impairment in patients with SSc, but by evaluating the endothelium-dependent flow-mediated dilation at the level of the brachial artery, and the impairment was already present in patients with early NC pattern vs controls [8]. Rosato et al. did not observe any association between DU history and US findings. On the contrary, in the study of Lüders et al. DUs/PSs were predominantly found in the same fingers that presented the highest percentages of pathological—and especially occluded—arteries. The findings are partially in contrast to the observations made by Rosato et al. since they are just as possible the result of a reduced comparability due to differences in arterial grading and the additional fingerwise evaluation made by Lüders et al. Unfortunately, in the study of Lüders et al. no healthy controls were considered, and no quantitative analysis was added, but just a qualitative and semi-quantitative evaluation (percentage) of the hand macrovasculature alterations. In addition, in Rosato et al.’s study patients were untreated at the time of the examinations, whereas in Lüders et al.’s trial, all concomitant treatments were allowed. As a matter of fact, Lüders et al. admitted as one of their study limitations to have included different therapies and