Cell Therapy for Critical Limb Ischemia: Advantages, Limitations, and New Perspectives for Treatment of Patients with Critical Diabetic Vasculopathy.

PURPOSE OF REVIEW: To provide a highlight of the current state of cell therapy for the treatment of critical limb ischemia in patients with diabetes. RECENT FINDINGS: The global incidence of diabetes is constantly growing with consequent challenges for healthcare systems worldwide. In the UK only, NHS costs attributed to diabetic complications, such as peripheral vascular disease, amputation, blindness, renal failure, and stroke, average £10 billion each year, with cost pressure being estimated to get worse. Although giant leaps forward have been registered in the scope of early diagnosis and optimal glycaemic control, an effective treatment for critical limb ischemia is still lacking. The present review aims to provide an update of the ongoing work in the field of regenerative medicine. Recent advancements but also limitations imposed by diabetes on the potential of the approach are addressed. In particular, the review focuses on the perturbation of non-coding RNA networks in progenitor cells and the possibility of using emerging knowledge on molecular mechanisms to design refined protocols for personalized therapy. The field of cell therapy showed rapid progress but has limitations. Significant advances are foreseen in the upcoming years thanks to a better understanding of molecular bottlenecks associated with the metabolic disorders.

Overexpression of miR-210 and its significance in ischemic tissue damage.

Hypoxia-induced miR-210 displays a pro-survival, cytoprotective and pro-angiogenic role in several in vitro systems. In vivo, we previously found that miR-210 inhibition increases ischemic damage. Here we describe the generation of a versatile transgenic mouse model allowing the evaluation of miR-210 therapeutic potential in ischemic cardiovascular diseases. We generated a Tet-On miR-210 transgenic mouse strain (TG-210) by targeted transgenesis in the ROSA26 locus. To functionally validate miR-210 transgenic mice, hindlimb ischemia was induced by femoral artery dissection. Blood perfusion was evaluated by power Doppler while tissue damage and inflammation were assessed by histological evaluation. We found that miR-210 levels were rapidly increased in TG-210 mice upon doxycycline administration. miR-210 overexpression was maintained over time and remained within physiological levels in multiple tissues. When hindlimb ischemia was induced, miR-210 overexpression protected from both muscular and vascular ischemic damage, decreased inflammatory cells density and allowed to maintain a better calf perfusion. In conclusion, we generated and functionally validated a miR-210 transgenic mouse model. Albeit validated in the context of a specific cardiovascular ischemic disease, miR-210 transgenic mice may also represent a useful model to assess the function of miR-210 in other physio-pathological conditions.

Contribution of pericyte paracrine regulation of the endothelium to angiogenesis.

During physiological development and after a stressor event, vascular cells communicate with each other to evoke new vessel formation-a process known as angiogenesis. This communication occurs via direct contact and via paracrine release of proteins and nucleic acids, both in a free form or encapsulated into micro-vesicles. In diseases with an altered angiogenic response, such as cancer and diabetic vascular complications, it becomes of paramount importance to tune the cell communication process. Endothelial cell growth and migration are essential processes for new vessel formation, and pericytes, together with some classes of circulating monocytes, are important endothelial regulators. The interaction between pericytes and the endothelium is facilitated by their anatomical apposition, which involves endothelial cells and pericytes sharing the same basement membrane. However, the role of pericytes is not fully understood. The characteristics and the function of tissue-specific pericytesis are the focus of this review. Factors involved in the cross-talk between these cell types and the opportunities afforded by micro-RNA and micro-vesicle techniques are discussed. Targeting these mechanisms in pathological conditions, in which the vessel response is altered, is considered in relation to identification of new therapies for restoring the blood flow.

Design, fabrication and perivascular implantation of bioactive scaffolds engineered with human adventitial progenitor cells for stimulation of arteriogenesis in peripheral ischemia.

Cell therapy represents a promising option for revascularization of ischemic tissues. However, injection of dispersed cells is not optimal to ensure precise homing into the recipient's vasculature. Implantation of cell-engineered scaffolds around the occluded artery may obviate these limitations. Here, we employed the synthetic polymer polycaprolactone for fabrication of 3D woodpile- or channel-shaped scaffolds by a computer-assisted writing system (pressure assisted micro-syringe square), followed by deposition of gelatin (GL) nanofibers by electro-spinning. Scaffolds were then cross-linked with natural (genipin, GP) or synthetic (3-glycidyloxy-propyl-trimethoxy-silane, GPTMS) agents to improve mechanical properties and durability in vivo. The composite scaffolds were next fixed by crown inserts in each well of a multi-well plate and seeded with adventitial progenitor cells (APCs, 3 cell lines in duplicate), which were isolated/expanded from human saphenous vein surgical leftovers. Cell density, alignment, proliferation and viability were assessed 1 week later. Data from in vitro assays showed channel-shaped/GPTMS-crosslinked scaffolds confer APCs with best alignment and survival/growth characteristics. Based on these results, channel-shaped/GPTMS-crosslinked scaffolds with or without APCs were implanted around the femoral artery of mice with unilateral limb ischemia. Perivascular implantation of scaffolds accelerated limb blood flow recovery, as assessed by laser Doppler or fluorescent microspheres, and increased arterial collaterals around the femoral artery and in limb muscles compared with non-implanted controls. Blood flow recovery and perivascular arteriogenesis were additionally incremented by APC-engineered scaffolds. In conclusion, perivascular application of human APC-engineered scaffolds may represent a novel option for targeted delivery of therapeutic cells in patients with critical limb ischemia.

Nitric oxide-donating statin improves multiple functions of circulating angiogenic cells.

BACKGROUND AND PURPOSE: Statins, a major component of the prevention of cardiovascular disease, aid progenitor cell functions in vivo and in vitro. Statins bearing a NO-releasing moiety were developed for their enhanced anti-inflammatory/anti-thrombotic properties. Here, we investigated if the NO-donating atorvastatin (NCX 547) improved the functions of circulating angiogenic cells (CACs). EXPERIMENTAL APPROACH: Circulating angiogenic cells (CACs) were prepared from peripheral blood monocytes of healthy volunteers and type-2 diabetic patients and were cultured in low (LG) or high glucose (HG) conditions, in presence of atorvastatin or NCX 547 (both at 0.1 µM) or vehicle. Functional assays (outgrowth, proliferation, viability, senescence and apoptosis) were performed in presence of the endothelial NOS inhibitor L-NIO, the NO scavenger c-PTIO or vehicle. KEY RESULTS: Culturing in HG conditions lowered NO in CACs, inhibited outgrowth, proliferation, viability and migration, and induced cell senescence and apoptosis. NCX 547 fully restored NO levels and functions of HG-cultured CACs, while atorvastatin prevented only apoptosis in CACs. The activity of Akt, a pro-survival kinase, was increased by atorvastatin in LG-cultured but not in HG-cultured CACs, whereas NCX 547 increased Akt activity in both conditions. L-NIO partially blunted and c-PTIO prevented NCX 547-induced improvements in CAC functions. Finally, NCX 547 improved outgrowth and migration of CACs prepared from patients with type 2 diabetes. CONCLUSIONS AND IMPLICATIONS: NCX 547 was more effective than atorvastatin in preserving functions of CACs. This property adds to the spectrum of favourable actions that would make NO-releasing statins more effective agents for treating cardiovascular disease.

Role of human tissue kallikrein in gastrointestinal stromal tumour invasion.

BACKGROUND: Human tissue kallikrein (hK1) generates vasodilator kinins from kininogen and promotes angiogenesis by kinin-dependent and kinin-independent mechanisms. Here, we investigate the expression and functional relevance of hK1 in human gastrointestinal stromal tumour (GIST). METHODS: Vascularisation and hK1 expression of GIST samples were assessed by immunohistochemistry. In two GIST cell lines, hK1 expression was assessed by PCR, and hK1 protein levels and activity were measured by ELISA and an amidolytic assay, respectively. The effect of hK1 silencing, inhibition or overexpression on GIST cell proliferation, migration and paracrine induction of angiogenesis was studied. Finally, local and systemic levels of hK1 were assessed in mice injected with GIST cells. RESULTS: Human tissue kallikrein was detected in 19 out of 22 human GIST samples. Moreover, GIST cells express and secrete active hK1. Titration of hK1 demonstrated its involvement in GIST invasive behaviour, but not proliferation. Furthermore, hK1 released by GIST cells promoted endothelial cell migration and network formation through kinin-dependent mechanisms. Gastrointestinal stromal tumour implantation in nude mice resulted in local and systemic hK1 expression proportional to tumour dimension. CONCLUSIONS: Human tissue kallikrein is produced and released by GIST and participates in tumour invasion. Further studies are needed to validate hK1 as a diagnostic biomarker and therapeutic target in GIST.

Adenovirus-mediated VEGF(165) gene transfer enhances wound healing by promoting angiogenesis in CD1 diabetic mice.

It has been previously shown that vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis during wound repair and that healing-impaired diabetic mice show decreased VEGF expression levels. In order to investigate the potential benefits of gene therapy with growth factors on wound repair, a replication-deficient recombinant adenovirus vector carrying the human VEGF(165) gene (AdCMV.VEGF(165)) was topically applied on excisional wounds of streptozotocin-induced diabetic mice. Treatment with AdCMV.VEGF(165) significantly accelerated wound closure when compared with AdCMV.LacZ-treated, as well as saline-treated control mice, by promoting angiogenesis at the site of injury. Our findings suggest that AdCMV.VEGF(165) may be regarded as a therapeutic tool for the treatment of diabetic ulcers.

The chemokine CXCL13 (BCA-1) inhibits FGF-2 effects on endothelial cells.

Several chemokines, belonging to both the CXC and CC classes, act as positive or negative regulators of angiogenesis. We sought to investigate the role of CXCL13, B cell-attracting chemokine 1 (BCA-1), also known as B-lymphocyte chemoattractant (BLC), on endothelial cell functions. We tested the effect of CXCL13 on HUVEC chemotaxis and proliferation in the presence of fibroblast growth factor (FGF)-2 and found that such chemokine inhibits FGF-2-induced functions, while is not active by itself. To test whether other FGF-2-mediated biological activities may be affected, we evaluated the ability of CXCL13 to rescue HUVEC from starvation-induced apoptosis, as FGF-2 is a survival factor for endothelial cells, and found that CXCL13 partially inhibits such rescue. Multiple mechanisms may be responsible for these biological activities as CXCL13 displaces FGF-2 binding to endothelial cells, inhibits FGF-2 homodimerization, and induces the formation of CXCL13-FGF-2 heterodimers. Our data suggest that CXCL13 may modulate angiogenesis by interfering with FGF-2 activity.

I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo.

Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription- polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).

Patterns of recurrence in patients with squamous cell carcinoma of the vulva. A multicenter CTF Study.

BACKGROUND: Invasive vulvar carcinoma is a rare disease with an incidence rate of 3-5% of all female genital neoplasms. The current study discusses the limited number of articles in the literature regarding the patterns of recurrence as well as the clinical outcome of patients with recurrent disease based on a consistent and consecutive series of cases. METHODS: A common clinical chart focusing on the study of patterns of recurrence was used in five Italian gynecologic institutions with uniform criteria of surgical nomenclature, pathologic variables, and sites of recurrence. Between 1980-1994, 502 cases of primary invasive squamous carcinoma of the vulva were registered consecutively, treated, and considered for this multicentered study. RESULTS: Of 502 patients, 187 (37.3%) developed a recurrence. Distribution of the recurrences by site was as follows: perineal, 53.4%; inguinal, 18.7%; pelvic, 5.7%; distant, 7.9%; and multiple, 14.2%. In a multivariate analysis, 3 characteristics appeared to be statistically correlated with the risk of recurrence: International Federation of Gynecology and Obstetrics Stage > II (P = 0.029), positive lymph nodes (P = 0.009), and vascular space invasion (P = 0.004). The 5-year survival rate was 60% for perineal recurrences, 27% for inguinal and pelvic recurrences, 15% for distant recurrences, and 14% for multiple recurrences. CONCLUSIONS: In the current study the prognostic factors found to have statistical significance as prognostic factors for risk of recurrence were tumor dimension, lymph node involvement, and stromal and vascular space invasion. The presence of inguinal lymph node metastases was predictive of multiple and distant recurrences with a low rate of incidence of isolated perineal recurrence (27%) compared with negative lymph node cases (57.5%). Survival analysis of recurrent disease showed that the surgical resection of local recurrences may provide acceptable results (51% at 5 years). This observation may justify a follow-up program aimed at identifying those patients with early local recurrence suitable for radical resection.

The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor.

Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional ligand for the EHV-2 E1 receptor. Chemokines are likely to play a role in the regulation of immune functions in equine hosts during EHV-2 infection and, via interaction with E1, may affect viral replication and/or escape from immune responses.

Identification of the CC chemokines TARC and macrophage inflammatory protein-1 beta as novel functional ligands for the CCR8 receptor.

Chemokines are key molecules in directing leukocyte migration toward sites of inflammation. We have previously cloned a putative CC chemokine receptor gene, TER1, whose expression is restricted to lymphoid tissues and cell lines. Recently, this receptor has been shown to signal in response to the human CC chemokine I-309 and thus it has been renamed CCR8 according to the current nomenclature. In the present study, we report the identification of the CC chemokines thymus and activation-regulated cytokine (TARC) and macrophage inflammatory protein-1 beta (MIP-1 beta) as CCR8 ligands, as they induce chemotaxis in CCR8 Jurkat stable transfectants. Furthermore, we have generated a polyclonal antiserum that is able to recognize the CCR8 molecule in transfectant lysates. The pattern of CCR8 mRNA expression and the functional effects exerted by its ligand suggest that the triggering of this receptor may regulate multiple functions including activation, migration and proliferation of lymphoid cells.

Molecular cloning of TER1, a chemokine receptor-like gene expressed by lymphoid tissues.

Several chemokine receptors have been cloned and shown to belong to a superfamily of seven transmembrane, G protein-coupled receptors. We report here the molecular cloning of TER1, a novel human chemokine receptor-like gene. The amino acid sequence deduced from the TER1 cDNA shows 43, 40, 40, and 39% identity to CCR4, CCR5, CCR1, and CCR2B beta chemokine receptors, respectively. By the use of fluorescent in situ hybridization, we have mapped the TER1 gene to chromosome 3p21, clustered with other chemokine receptor genes. By Northern blot analysis, TER1 mRNA is found to be expressed in the thymus, spleen, and at barely detectable levels in peripheral blood lymphocytes. Moreover, TER1 message in abundant in the NK cell line NK3.3 and in the T cell line MOLT-4. The restricted TER1 expression in cells and tissues of the lymphoid lineage suggests that this receptor may play a role in regulating immune functions.