Accessory cells for ß-cell transplantation.

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous ß-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive ß-cell regeneration remain enigmatic and ß-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in ß-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

Human blood outgrowth endothelial cells improve islet survival and function when co-transplanted in a mouse model of diabetes.

AIMS/HYPOTHESIS: As current islet-transplantation protocols suffer from significant graft loss and dysfunction, strategies to sustain the long-term benefits of this therapy are required. Rapid and adequate oxygen and nutrient delivery by blood vessels improves islet engraftment and function. The present report evaluated a potentially beneficial effect of adult human blood outgrowth endothelial cells (BOEC) on islet graft vascularisation and function. METHODS: Human BOEC, 5 × 10(5), were co-transplanted with a rat marginal-islet graft under the kidney capsule of hyperglycaemic NOD severe combined immunodeficiency (SCID) mice, and the effect on metabolic outcome was evaluated. RESULTS: Although vessel density remained unaffected, co-transplantation of islets with BOEC resulted in a significant and specific improvement of glycaemia and increased plasma C-peptide. Moreover, in contrast to control mice, BOEC recipients displayed reduced beta cell death and increases in body weight, beta cell proliferation and graft-vessel and beta cell volume. In vivo cell tracing demonstrated that BOEC remain at the site of transplantation and do not expand. The potential clinical applicability was underscored by the observed metabolic benefit of co-transplanting islets with BOEC derived from a type 1 diabetes patient. CONCLUSIONS/INTERPRETATION: The present data support the use of autologous BOEC in translational studies that aim to improve current islet-transplantation protocols for the treatment of brittle type 1 diabetes.

Plasminogen activation: a mediator of vascular smooth muscle cell apoptosis in atherosclerotic plaques.

BACKGROUND: Apoptosis of vascular cells is considered to be a major determinant of atherosclerotic plaque vulnerability and potential rupture. Plasmin can be generated in atherosclerotic plaques and recent in vitro data suggest that plasminogen activation may trigger vascular smooth muscle cell (VSMC) apoptosis. AIM: To determine whether plasminogen activation may induce aortic VSMC apoptosis ex vivo and in vivo. METHODS AND RESULTS: Mice with single or combined deficiencies of apolipoprotein E (ApoE) and plasminogen activator inhibitor-1 (PAI-1) were used. Ex vivo incubation with plasminogen of isolated aortic tunica media from PAI-1-deficient mice induced plasminogen activation and VSMC apoptosis, which was inhibited by alpha2-antiplasmin. In vivo, levels of plasmin, active caspase 3 and VSMC apoptotic index were significantly higher in atherosclerotic aortas from mice with combined ApoE and PAI-1 deficiencies than in those from littermates with single ApoE deficiency. A parallel decrease in VSMC density was observed. CONCLUSIONS: These data strongly suggest that plasminogen activation may contribute to VSMC apoptosis in atherosclerotic plaques.

Placental growth factor and its receptor, vascular endothelial growth factor receptor-1: novel targets for stimulation of ischemic tissue revascularization and inhibition of angiogenic and inflammatory disorders.

In contrast to VEGF and its receptor VEGFR-2, PlGF and its receptor VEGFR-1 have been largely neglected and therefore their potential for therapy has not been previously explored. In this review, we describe the molecular properties of PlGF and VEGFR-1 and how this translates into an important role for PlGF in the angiogenic switch in pathological angiogenesis, by interacting with VEGFR-1 and synergizing with VEGF. PlGF was effective in the growth of new and stable vessels in cardiac and limb ischemia, through its action on different cell types (i.e. endothelial, smooth muscle and inflammatory cells and their precursors) that play a cardinal role in blood vessel formation. Accordingly, blocking its receptor VEGFR-1 with monoclonal antibodies (anti-VEGFR-1 mAb), expressed on al these cell types, successfully attenuated blood vessel formation during cancer, ischemic retinopathy and rheumatoid arthritis. In addition, while blocking this receptor was effective in reducing inflammatory disorders like atherosclerosis and rheumatoid arthritis, blocking the anti-angiogenic receptor VEGFR-2 was without effect. This indicates that in the latter diseases the beneficial effects of anti-VEGFR1 mAb were mainly due to its effect on inflammatory cells. Importantly, VEGFR-1 was also present on hematopoietic stem/progenitor cells, the precursors of inflammatory cells. Thus, these preclinical studies show proof-of-principle that PlGF and VEGFR-1 are promising therapeutic targets to treat angiogenesis and inflammation related disorders. Clinical trials will reveal whether this is also true for patients.

Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells.

BACKGROUND: Monocytes may have an important role in the resolution of venous thrombosis. Increased expression of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) is associated with an ingress of monocytes into the thrombus. This study was designed to evaluate the importance of these activators in thrombus resolution. METHODS AND RESULTS: Inferior caval vein thrombosis was induced in cohorts of adult wild-type, uPA gene-knockout (uPA-/-), and tPA gene-knockout (tPA-/-) mice in a flow model. Thrombi were harvested from wild-type and uPA-/- mice (n=60 per group) between 1 and 60 days. Thrombi were also obtained from groups of wild-type and tPA-/- mice (n=24 per group) between 1 and 28 days. Thrombus size and macrophage content were measured by computer-assisted image analysis. Thrombus resolution was significantly impaired in the uPA-/- mice compared with wild-type controls (P<0.0001) but was unaffected in tPA-/- mice. Monocyte content in wild-type mice was highest at 14 days after thrombus induction and was approximately 4 times greater than in uPA-/- mice (P=0.0043). Thrombus size in uPA-/- mice transplanted with wild-type marrow (0.29+/-0.06 mm2) was significantly smaller than in uPA-/- mice given uPA-/- bone marrow (3.9+/-1.1 mm2) (P=0.0022). Donor bone marrow-derived cells expressing LacZ were present in the thrombus after transplantation. CONCLUSIONS: The resolution of experimental venous thrombus is dependent on uPA but is unaffected by the absence of tPA. Absence of uPA is also associated with delayed monocyte recruitment into the thrombus. Transplanting wild-type bone marrow restores thrombus resolution in uPA-/- animals, suggesting an important role for bone marrow-derived cells in this process.

The emerging role of the bone marrow-derived stem cells in (therapeutic) angiogenesis.

Proper formation of blood vessels (angiogenesis) is essential for development, reproduction and wound healing. When derailed, angionenesis contributes to numerous lifethreatening disorders. While research has generally been focusing on the two main vascular cell types (endothelial and smooth muscle cells), recent evidence indicates that bone marrow may also contribute to this process, both in the embryo and the adult. Novel vascular progenitors, even one common to both endothelial and smooth muscle cells, have been identified in the embryo. An exciting observation is that endothelial precursors have also been identified in the adult bone marrow. Transplantation studies revealed that these precursors as well as other bone marrow-derived cells contribute to the growth of endothelium-lined vessels (angiogenesis) as well as the expansion of pre-existing collaterals (arteriogenesis) in ischemic disease. These findings have raised hopes that bone marrow-derived cells might one day become useful for cell-based angiogenic therapy.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Genetic studies on the role of proteinases and growth factors in atherosclerosis and aneurysm formation.

Expression analysis and epidemiologic studies have provided indirect evidence that proteinases and growth factors play a role in the development of atherosclerosis and complications such as aneurysm formation and plaque rupture. Studies using genetically altered mice have proven to be an elegant tool to study the causal involvement of these factors in atherogenesis and to gain insight into the underlying mechanisms. Recently, proteinases of the plasminogen and matrix metalloproteinase (MMP) systems as well as their inhibitors have received much attention, and these studies together have emphasized the complexity of their role in vascular disease. This overview summarizes the current knowledge on plasminogen activator inhibitor-1 (PAI-1) in the progression of atherosclerosis and the influence of MMPs in aneurysm formation. In addition, a possible role for Gas6, the product of growth arrest-specific gene 6, in atherosclerotic lesion development is put into perspective.

The role of proteinases in angiogenesis, heart development, restenosis, atherosclerosis, myocardial ischemia, and stroke: insights from genetic studies.

The development of novel gene technologies in mice has provided an elegant tool to identify gene products that are causally linked to certain physiologic processes as well as the pathogenesis of numerous disorders. Using these techniques, three major proteolytic systems -- the plasminogen, the matrix metalloproteinase (MMP) and the coagulation systems -- have been shown to be involved in cardiovascular diseases, which still constitute the leading cause of death in Western societies. This overview summarizes the role of these proteolytic systems in angiogenesis, arterial stenosis, allograft transplant stenosis, vein graft stenosis, atherosclerosis, myocardial infarction, cardiac development and ischemic stroke and discusses possible therapeutic implications.

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.