Microaneurysm turnover in diabetic retinopathy assessed by automated RetmarkerDR image analysis--potential role as biomarker of response to ranibizumab treatment.

PURPOSE: To evaluate the influence of a ranibizumab treatment on microaneurysm (MA) turnover in diabetic retinopathy. METHODS: Sixty-nine eyes were included in this retrospective study. We compared a group of 33 eyes with ranibizumab treatment for diabetic macular edema to 36 eyes with nonproliferative diabetic retinopathy only. Nonmydriatic ultra-widefield scanning laser ophthalmoscopy (Optomap) images were obtained at a mean 4.76 ± 1.69 days prior to the first ranibizumab injection (baseline) and again 35.94 ± 2.44 days after the third consecutive injection in a 4-week interval. In untreated controls, images were obtained at baseline and 97.81 ± 3.16 days thereafter. Images were analyzed using the RetmarkerDR software (Critical Health SA, Coimbra, Portugal), and the turnover of MAs was documented and analyzed. Thereafter, MA turnover was correlated with central retinal thickness (CRT) as assessed by OCT. RESULTS: At baseline, patients in the treatment group had 5.64 ± 0.75 MAs. One month after 3 ranibizumab injections, measured MAs decreased to 4.03 ± 0.66. In the untreated control group, the initial number of 3.36 ± 0.6 MAs remained almost unchanged over 3-4 months (2.89 ± 0.57 MAs). Dynamic analysis showed that after ranibizumab treatment 3.06 ± 0.5 new MAs appeared, while 5.09 ± 0.79 disappeared. In the control group, 2.11 ± 0.4 new MAs appeared and 2.61 ± 0.48 disappeared. MA turnover was significantly higher with ranibizumab compared to the control group (8.15 ± 1.14 vs. 4.72 ± 0.81, p < 0.001). Consistently, CRT decreased from 444 to 330 µm in the ranibizumab group, while there was no change in the control group (291 vs. 288 µm). CONCLUSION: The treatment of macular edema using ranibizumab does not only reduce macular thickness, but also has an impact on the turnover of MAs in diabetic retinopathy. RetmarkerDR analysis showed that more pre-existent MAs disappeared than new MAs developed, and the absolute number of MAs also decreased.

Vascular incorporation of endothelial colony-forming cells is essential for functional recovery of murine ischemic tissue following cell therapy.

OBJECTIVE: Cord blood-derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell-based therapy, including mesenchymal stem cells (MSCs). METHODS AND RESULTS: ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor(+) cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. CONCLUSIONS: Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell-based therapy represents a promising approach for vascular regeneration of ischemic tissue.

Adiponectin pretreatment counteracts the detrimental effect of a diabetic environment on endothelial progenitors.

OBJECTIVE: It has been shown that vascular progenitors from patients with diabetes are dysfunctional. However, therapeutic strategies to counteract their reduced functional capacity are still lacking. Because adiponectin has reported salutary effects on endothelial function, we investigated the functional effects of globular adiponectin (gAcrp), the active domain of adiponectin, on isolated endothelial colony-forming cells (ECFC). RESEARCH DESIGN AND METHODS: ECFC were isolated from peripheral blood of type 2 diabetic patients (dmECFC) and compared with ECFC of healthy young volunteers (yECFC) and nondiabetic age-matched control subjects (hECFC). Cells were treated with gAcrp for 48 h followed by assessment of cell counts, cell cycle analysis, and migration capacity. For in vivo evaluation, human ECFC were injected into normoglycemic or streptozotocin-induced hyperglycemic nu/nu mice after hind limb ischemia. RESULTS: Whereas dmECFC were functionally impaired compared with yECFC and hECFC, gAcrp significantly enhanced their in vitro proliferation and migratory activity. In vitro effects were significantly stronger in hECFC compared with dmECFC and were mediated through the cyclooxygenase-2 pathway. Most important, however, we observed a profound and sustained increase of the in vivo neovascularization in mice receiving gAcrp-pretreated dmECFC compared with untreated dmECFC under both normoglycemic and hyperglycemic conditions. CONCLUSIONS: Pretreatment of ECFC with gAcrp enhanced the functional capacity of ECFC in vitro and in vivo in normoglycemic and hyperglycemic environments. Therefore, preconditioning of dmECFC with gAcrp may be a novel approach to counteract their functional impairment in diabetes.

Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization.

OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer.

BACKGROUND & AIMS: Pancreatic cancers contain exclusively tumorigenic cancer stem cells (CSCs), which are highly resistant to chemotherapy, resulting in a relative increase in CSC numbers during gemcitabine treatment. Signaling through sonic hedgehog and mammalian target of rapamycin (mTOR), respectively, may be essential for CSC self-renewal and could represent putative targets for novel treatment modalities. METHODS: We used in vitro and in vivo models of pancreatic cancer to examine the effects of sonic hedgehog inhibition (cyclopamine/CUR199691) and mTOR blockade (rapamycin) on the tumorigenic CSC population. RESULTS: Surprisingly, neither cyclopamine nor rapamycin alone or as supplements to chemotherapy were capable of effectively diminishing the CSC pool. Only the combined inhibition of both pathways together with chemotherapy reduced the number of CSCs to virtually undetectable levels in vitro and in vivo. Most importantly, in vivo administration of this triple combination in mice with established patient-derived pancreatic tumors was reasonably tolerated and translated into significantly prolonged long-term survival. CONCLUSIONS: The combined blockade of sonic hedgehog and mTOR signaling together with standard chemotherapy is capable of eliminating pancreatic CSCs. Further preclinical investigation of this promising approach may lead to the development of a novel therapeutic strategy to improve the devastating prognosis of patients with pancreatic cancer.

Prostaglandin E positively modulates endothelial progenitor cell homeostasis: an advanced treatment modality for autologous cell therapy.

AIMS: The mobilization of endothelial progenitor cells (EPC) and their functioning in postnatal neovascularization are tightly regulated. To identify new modulators of EPC homeostasis, we screened biologically active prostaglandin E compounds for their effects on EPC production, trafficking and function. METHODS AND RESULTS: We found that EPC are a rich source for prostaglandin E(2) (PGE(2)), stimulating their number and function in an auto- and paracrine manner. In vivo blockade of PGE(2) production by selective cyclooxygenase-2 inhibition virtually abrogated ischemia-induced EPC mobilization demonstrating its crucial role in EPC homeostasis following tissue ischemia. Conversely, ex vivo treatment of isolated EPC with the clinically approved PGE(1) analogue alprostadil enhanced EPC number and function. These effects were mediated by increased expression of the chemokine receptor CXCR4 and were dependent on nitric oxide synthase activity. Most importantly, ex vivo PGE(1) pretreatment of isolated EPC significantly enhanced their neovascularization capacity in a murine model of hind limb ischemia as assessed by laser Doppler analysis, exercise stress test and immunohistochemistry. CONCLUSIONS: The conserved role for PGE in the regulation of EPC homeostasis suggests that ex vivo modulation of the prostaglandin pathway in isolated progenitor cells may represent a novel and safe strategy to facilitate cell-based therapies.