Marina Cinelli

Autologous stem cell transplantation improves microcirculation in systemic sclerosis

Background: In systemic sclerosis (SSc) reduced capillary density decreases blood flow and leads to tissue ischaemia and fingertip ulcers. Nail fold videocapillaroscopy (NVC) is a diagnostic and follow-up parameter useful to evaluate the severity, activity and the stage of SSc microvascular damage. Autologous haemopoietic stem cell transplantation (HSCT) is a new treatment for patients with severe diffuse cutaneous systemic sclerosis (dcSSc) refractory to conventional therapies. We aimed to evaluate the improvement of microvasculature after HSCT using NVC. Methods: A total of 16 patients with severe dcSSc with a “late” videocapillaroscopy pattern underwent an immunesuppressive treatment: 6 were treated with HSCT and 10 with monthly pulse cyclophosphamide (CYC) 1 g for 6 months and then orally with 50 mg/day for further 6 months. NVC was performed before and after 3 months from the beginning of each treatment and then repeated every 3 months. Results: In all patients, before HSCT NVC showed large avascular areas and ramified capillaries and vascular architectural disorganisation (“late” pattern). At 3 months after HSCT, the NVC pattern changed from “late” into “active”, showing frequent giant capillaries (>6/mm) and haemorrhages, absence of avascular areas and angiogenesis phenomena; 1 year after HSCT, microvascular abnormalities were still in the “active” pattern. In patients treated with CYC, no NVC modifications were observed during 24 months of follow-up and the pattern always remained “late”. Conclusions: These results indicate that HSCT with a high dose CYC regimen may foster vascular remodelling, while CYC at lower doses and with a chronic regimen does not influence the microvasculature.

Bosentan regulates the expression of adhesion molecules on circulating T cells and serum soluble adhesion molecules in systemic sclerosis-associated pulmonary arterial hypertension

Objectives: To study the expression of adhesion molecules in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and the effects of therapy with the endothelin-1 (ET-1) receptor antagonist, bosentan. Methods: In all, 35 patients with SSc and 25 healthy donors (HD) were selected for this study. Of 35 patients, 10 had isolated PAH assessed by Doppler echocardiography and treated with bosentan. Peripheral blood (PB) lymphocytes were isolated by density gradient centrifugation, and the expression of lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA-4) and L-selectin on CD3 T cells was assessed by double immunofluorescence and flow-cytometry. As endothelial activation markers, serum soluble P-selectin, platelet/endothelial cell adhesion molecule (PECAM)-1, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1 and von Willebrand factor (vWF) antigen were assessed by ELISA. In patients with SSc-PAH, T cell subsets and soluble endothelial markers were assessed at baseline and after 6 and 12 months of bosentan therapy. Results: In patients with SSc-PAH, serum soluble ICAM-1, VCAM-1, P-selectin and PECAM-1 levels were higher than in HD at baseline and fell to normal values after 12 months of bosentan therapy. CD3–LFA1 T cells were significantly higher in PAH-SSc at baseline than in HD or SSc and significantly decreased after therapy. CD3–L-selectin T cells were significantly lower in SSc-PAH at baseline than in HD or SSc and rose to normal levels after bosentan therapy. Conclusions: This study confirms that endothelial activation occurs in SSc, and suggests that changes in the T cell/endothelium interplay take place in SSc-associated PAH. Bosentan seems to be able to hamper these changes and restore T cell functions in these patients.

Systemic sclerosis fibroblasts inhibit in vitro angiogenesis by MMP-12-dependent cleavage of the endothelial cell urokinase receptor

Failure of endothelial cells to develop new vessels in response to hypoxia is a distinctive feature of systemic sclerosis (SSc) in the avascular phase. We have previously shown that SSc endothelial cells over‐express matrix metalloproteinase‐12 (MMP‐12), which blocks angiogenesis by cleavage of the endothelial urokinase‐type plasminogen activator receptor (uPAR). In the present study, we have investigated whether over‐expression of MMP‐12 and of angiostatic factors, or hypo‐expression of angiogenic factors by SSc fibroblasts, contributes to impaired angiogenesis in SSc. Dermal fibroblasts were isolated from healthy subjects (N‐Fb) and patients with diffuse SSc (SSc‐Fb). Angiogenesis of target normal human microvascular endothelial cells (H‐MVECs) was assayed by Matrigel invasion, cell proliferation, and capillary morphogenesis. uPAR cleavage and MMP‐12 activity were evaluated by western blotting. We show that the over‐expression of MMP‐12 by SSc‐Fb determines uPAR cleavage in H‐MVECs. Conditioned medium from SSc‐Fb impaired H‐MVEC proliferation, invasion, and capillary morphogenesis. Anti‐MMP‐12 antibodies restored such impairment. Altered expression of angiostatic/angiogenic factors, including transforming growth factor β1, did not account for SSc‐Fb‐dependent impairment of angiogenesis. The over‐expression of MMP‐12 by both SSc‐Fb and SSc endothelial cells indicates that MMP‐12 over‐production may have a critical pathogenic role in SSc‐associated vascular alterations. Copyright

Exercise Doppler Echocardiography Identifies Preclinic Asymptomatic Pulmonary Hypertension in Systemic Sclerosis

Abstract:  In systemic sclerosis (SSc), the involvement of the interstitium or vascular system of the lung may lead to pulmonary arterial hypertension (PAH). PAH is often asymptomatic or oligosymptomatic in early SSc and, when it becomes symptomatic, pulmonary vascular system is already damaged. Exercise echocardiography (ex‐echo), measuring pulmonary artery pressure (PAP) during exercise and allowing to differentiate physiologic from altered PAP responses, may identify subclinical PAH. Our aims were (a) to evaluate by ex‐echo the change of PAP in patients with SSc without lung involvement; and (b) to correlate PAP during exercise (ex‐PAP) values to clinical and biohumoral parameters of PAH. Twenty‐seven patients with limited SSc (lSSc) without interstitial lung involvement were studied. Patients underwent rest and exercise two‐dimensional and Doppler echocardiography by supine cycloergometer. Systolic PAP was calculated using the maximum systolic velocity of the tricuspid regurgitant jet at rest and during exercise values of systolic PAP exceeding 40 mmHg at ex‐echo were considered as abnormal, and biohumoral markers potentially related to PAH were assessed. Eighteen of 27 SSc patients presented an ex‐PAP >40 mmHg, while in 9 of 27 patients ex‐PAP values remained <40 mmHg (48.8 ± 4.5 mmHg versus 36.2 ± 3.1 mmHg; P < 0.001). Other echocardiographic and ergometric parameters, clinical tests, and biohumoral markers were not different in the two groups. Ex‐PAP significantly correlated with D‐dimer (P= 0.0125; r2= 0.2029). Ex‐echo identifies a cluster of SSc patients with subclinical PAH that may develop PAH. This group should be followed up and may be considered for specific therapies to prevent disease evolution.

Reduced circulating levels of angiotensin-(1–7) in systemic sclerosis: a new pathway in the dysregulation of endothelial-dependent vascular tone control

Objective: Systemic sclerosis (SSc) impairs endothelium-dependent vasodilatation. Among angiotensin I (Ang I)-derived compounds, vasoconstrictor angiotensin II (Ang II) and vasodilator angiotensin-(1–7) (Ang-(1–7)), cleaved from ACE and neutral endopeptidase (NEP) 24.11, respectively, play an important role in vascular tone regulation. Ang-(1–7) may act independently or by activating other vasodilating molecules, such as nitric oxide (NO) or prostaglandin I2 (PGI2). Our aim was to assess, in patients with SSc, circulating levels of Ang I, Ang II and Ang-(1–7), with their metabolising enzymes ACE and NEP, and levels of NO and PGI2, and to correlate them to the main characteristics of SSc. Methods: Levels of Ang I, Ang II, Ang-(1–7), NEP, ACE, NO and PGI2 were measured in 32 patients with SSc, who were also assessed for humoral and clinical characteristics, and 55 controls. Results: Plasma Ang I, Ang II and Ang-(1–7) levels were lower in patients with SSc than in controls (p<0.001in all cases). When Ang II and Ang-(1–7) levels were expressed as a function of the available Ang I, lower Ang-(1–7) levels in patients with SSc than in controls were confirmed (p<0.001), while no difference was found for Ang II levels. In patients with SSc, the Ang II/Ang-(1–7) ratio indicated a prevalence of Ang II over Ang-(1–7), while in controls Ang-(1–7) was prevalent (p<0.001). Levels of ACE, NEP, NO and PGI2 were lower in patients with SSc than in controls (p<0.05 in all cases). Conclusion: In patients with SSc, prevalence of the vasoconstricting Ang II over the vasodilator Ang-(1–7) suggests a dysfunction of the angiotensin-derived cascade that may contribute to dysregulation of vascular tone.

TNFα blockade prevents the development of inflammatory bowel disease in HLA‐B27 transgenic rats

Rats transgenic for HLA‐B27 and human β2microglobulin (B27TR) develop a multi‐systemic disease resembling inflammatory bowel disease (IBD) and spondyloarthritis. TNFα has a crucial role in chronic inflammation. Our objective was to evaluate the effect of anti‐TNFα treatment on spontaneous IBD in B27TR. Nine‐week‐old B27TR received monoclonal anti‐TNFα or an isotypic IgG2a,k up to age of 18 weeks. A second group was monitored up to 18 weeks and then randomly assigned to anti‐TNFα or IgG2 a,k treatment. Each rat was monitored for clinical IBD manifestations. After sacrifice, the colon was examined for pathological changes. TNFα receptors (TNF‐R1, TNF‐R2), Fas/Fas‐L expression and apoptosis were evaluated. IgG2a,k‐treated and untreated B27TR presented signs of IBD at 11 weeks, whereas in anti‐TNFα‐treated B27TR no IBD signs were detected. In the late treatment, IBD signs improved after 1 week. Histopathological analysis of IgG2a,k‐treated B27TR colon showed inflammatory signs that were widely prevented by early anti‐TNFα treatment. Late treatment did not significantly reduce inflammation. TNF‐R1 was weakly expressed in intestinal epithelial cells of IgG2a,k‐treated B27TR, while it was comparable to controls in anti‐TNFα‐treated animals. TNF‐R2 immunopositivity was strongly evident in IgG2a,k‐treated B27TR, whereas was absent in anti‐TNFα‐treated rats. RT‐PCR confirmed these results. IgG2a,k‐treated B27TR showed, at 18 weeks, few Fas‐positive cells and an increase of Fas‐L‐positive cells. At 27 weeks, Fas‐/Fas‐L‐positive cell number was significantly low. Anti‐TNFα treatment increased Fas‐L expression, whereas Fas increased only with the early treatment. TNFα blockade is effective in preventing inflammation in early phase of IBD, maintaining the homeostatic balance of apoptosis.

Piascledine modulates the production of VEGF and TIMP‐1 and reduces the invasiveness of rheumatoid arthritis synoviocytes

Background: In rheumatoid arthritis (RA), hypertrophy of the synovial membrane generates a tumour‐like pannus that invades the joint cavity and erodes cartilage and bone. Invasion of the extracellular matrix (ECM) is accompanied by angiogenesis, in which vascular endothelial growth factor (VEGF) and tissue inhibitors of metalloproteinases (TIMPs), produced by synoviocytes lining the pannus, have a primary role. Piascledine (PSD) is used in the treatment of osteoarthritis and has anti‐inflammatory effects in vitro. Objective: To study the effects of PSD on levels of VEGF and TIMP‐1 and chemoinvasion in RA synoviocytes and healthy controls. Methods: The effects of PSD 5, 10, and 20 µg/mL were evaluated, with/without interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNFα) 20 ng/mL, on synoviocytes. The levels of VEGF and TIMP‐1 were assayed in the culture medium by enzyme‐linked immunosorbent assay (ELISA). Chemoinvasion was measured by the Boyden chamber invasion assay. Results: RA synoviocytes treated with PSD showed, compared to basal, lower levels of VEGF (41080±830 vs. 79210±920 pg/106 cells, p<0.001) and increased levels of TIMP‐1 (23540±93.2 vs. 12860±42.9 ng/106 cells, p<0.001). PSD decreased dose‐dependently IL‐1β and TNFα induced migration. Conclusions: In RA synoviocytes, and also to a lesser extent in control cells, PSD modulates VEGF and TIMP‐1 and decreases chemoinvasion. PSD might have a role in the treatment of RA synovitis controlling invasiveness.

Angiotensin‐Converting Enzyme in Systemic Sclerosis

Abstract:  The main pathologic hallmark of systemic sclerosis (SSc) is endothelial derangement; the pathologic alterations of the vessel wall in SSc are strikingly similar to the modification detected in the atherosclerotic lesions, and it is now evident that SSc is also characterized by accelerated macrovascular disease. Peptides related to angiotensin II, the final product of the renin–angiotensin system (RAS), play a role as regulators of endothelial cell function. Angiotensin‐converting enzyme (ACE), the key enzyme in the RAS, is the predominant pathway of angiotensin II formation in blood and tissues. In intron 16 of the gene encoding for ACE an insertion/deletion (I/D) polymorphism, consisting of the presence or absence of a 287–base pair Alu sequence, has been identified. This polymorphism has been related to ACE enzyme levels, and data from experimental studies reported a functional role for this polymorphism in modulating the angiotensin II levels. We previously documented a high ACE D allele frequency in SSc patients and its role in increasing the risk of SSc, thus suggesting that the I/D polymorphism might be a useful genetic marker to identify SSc patients at risk to develop a severe vascular disease, frequently leading to gangrene. Moreover, our preliminary data, besides supporting the role of ACE I/D polymorphism as a predisposing factor to SSc, demonstrated its involvement in accelerated macrovascular disease by increasing the intima media thickness. Therefore, in SSc, not only endothelial dysfunction, but also vascular damage, linked to ACE I/D polymorphism, may significantly contribute to accelerated macrovascular disease, as the ACE D allele, by regulating both the production of angiotensin II and the degradation of bradykinin, contributes to mechanisms involved in the induction and maintenance of vessel wall modification.

Raloxifene reduces urokinase-type plasminogen activator-dependent proliferation of synoviocytes from patients with rheumatoid arthritis.

Extracellular fibrinolysis, controlled by the membrane-bound fibrinolytic system, is involved in cartilage damage and rheumatoid arthritis (RA) synovitis. Estrogen status and metabolism seem to be impaired in RA, and synoviocytes show receptors for estrogens. Our aims in this study were to evaluate in healthy and RA synoviocytes the effects of Raloxifene (RAL), a selective estrogen receptor modulator (SERM), on: proliferation; the components of the fibrinolytic system; and chemoinvasion. The effects of RAL were studied in vitro on synoviocytes from four RA patients and four controls. Proliferation was evaluated as cell number increase, and synoviocytes were treated with 0.5 μM and 1 μM RAL with and without urokinase-plasminogen activator (u-PA) and anti-u-PA/anti-u-PA receptor (u-PAR) antibodies. Fibrinolytic system components (u-PA, u-PAR and plasminogen activator inhibitor (PAI)-1) were assayed by ELISA with cells treated with 0.5 μM and 1 μM RAL for 48 h. u-PA activity was evaluated by zymography and a direct fibrinolytic assay. U-PAR/cell and its saturation were studied by radioiodination of u-PA and a u-PA binding assay. Chemoinvasion was measured using the Boyden chamber invasion assay. u-PA induced proliferation of RA synoviocytes was blocked by RAL (p < 0.05) and antagonized by antibodies alone. The inhibitory effect of RAL was not additive with u-PA/u-PAR antagonism. RA synoviocytes treated with RAL showed, compared to basal, higher levels of PAI-1 (10.75 ± 0.26 versus 5.5 ± 0.1 μg/106 cells, respectively; p < 0.01), lower levels of u-PA (1.04 ± 0.05 versus 3.1 ± 0.4 ng/106 cells, respectively; p < 0.001), and lower levels of u-PAR (11.28 ± 0.22 versus 23.6 ± 0.1 ng/106 cells, respectively; p < 0.001). RAL also significantly inhibited u-PA-induced migration. Similar effects were also shown, at least partially, in controls. RAL exerts anti-proliferative and anti-invasive effects on synoviocytes, mainly modulating u-PAR and, to a lesser extent, u-PA and PAI-1 levels, and inhibiting cell migration and proliferation.

Apoptosis: Molecular regulation of cell death and hematologic malignancies

We describe the molecular mechanisms of apoptosis and its relationships with hematologic malignancies, stressing the concept that, both positive and negative deregulation of apoptosis, may be involved in hematologic human diseases. So, this fundamental process must be balanced by so far unknown mechanisms, involving caspases (cysteine proteases, cleaving the protein substrate after an aspartate residue). These, so far known, ten proteases, are interconnected in a molecular cascade, initiated by the release of cytochrome C from mitochondrial membranes and its interaction with APAF-1 (the homolog of the Caenorhabditis e. CED-4) and with caspase 9, that initiates the proteolitic cascade (1,2). The conclusion is that apoptosis is a very important process, but yet poorly known in molecular details, in spite of the efforts of many scientists. Even the role of bcl-2, the main gene protecting from apoptosis, is still unknown. We close this chapter with a list of ten different technical approaches that can be useful tools to study apoptosis, and tracing the molecular principles on which they are based.