Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 1, 3 (RIPK1, RIPK3) and mixed lineage kinase domain-like protein (MLKL). The kinase of RIPK3 phosphorylates MLKL causing MLKL to form a pore-like structure, allowing intracellular contents to release and cell death to occur. Alternatively, RIPK1 and RIPK3 have been shown to regulate cytokine production directly influencing inflammatory immune infiltrates. Recent data suggest that necroptosis may contribute to the malignant transformation of tumor cells in vivo and we asked whether necroptosis may have a role in the tumor microenvironment altering the ability of the tumor to grow or metastasize. To determine if necroptosis in the tumor microenvironment could promote inflammation alone or by initiating necroptosis and thereby influencing growth or metastasis of tumors, we utilized a syngeneic tumor model of metastasis. Loss of RIPK3 in the tumor microenvironment reduced the number of tumor nodules in the lung by 46%. Loss of the kinase activity in RIPK1, a member of the necrosome also reduced tumor nodules in the lung by 38%. However, the loss of kinase activity in RIPK3 or the loss of MLKL only marginally altered the ability of tumor cells to form in the lung. Using bone marrow chimeras, the decrease in tumor nodules in the Ripk3−/− appeared to be due to the stromal compartment rather than the hematopoietic compartment. Transmigration assays showed decreased ability of tumor cells to transmigrate through the vascular endothelial layer, which correlated with decreased permeability in the Ripk3−/− mice after tumor injection. In response to permeability factors, such as vascular endothelial growth factor, RIPK3 null endothelial cells showed decreased p38/HSP27 activation. Taken together, our results suggest an alternative function for RIPK1/RIPK3 in vascular permeability leading to decreased number of metastasis.

SummaryHuman endothelial colony-forming cells (ECFCs) represent a promising source of adult stem cells for vascular repair, yet their regenerative capacity is limited. Here, we set out to understand the molecular mechanism restricting the repair function of ECFCs. We found that key pro-angiogenic pathways are repressed in ECFCs due to the presence of bivalent (H3K27me3/H3K4me3) epigenetic marks, which decreases the cells' regenerative potential. Importantly, ex vivo treatment with a combination of epigenetic drugs that resolves bivalent marks toward the transcriptionally active H3K4me3 state leads to the simultaneous activation of multiple pro-angiogenic signaling pathways (VEGFR, CXCR4, WNT, NOTCH, SHH). This in turn results in improved capacity of ECFCs to form capillary-like networks in vitro and in vivo. Furthermore, restoration of perfusion is accelerated upon transplantation of drug-treated ECFCs in a model of hindlimb ischemia. Thus, ex vivo treatment with epigenetic drugs increases the vascular repair properties of ECFCs through transient activation of pro-angiogenic signaling pathways.

Objective: The incorporation of high atomic number materials such as gold nanoparticles (GNPs) into tumor cells is being tested to enhance the local radiotherapy (RT) dose. It is also known that the radiosensitivity of tumor cells depends on the phase of their cell cycle. Triple combination of GNPs, phase of tumor cell population, and RT for improved outcomes in cancer treatment. Methods: We used a double-thymidine block method for synchronization of the tumor cell population. GNPs of diameters 17 and 46 nm were used to capture the size dependent effects. A radiation dose of 2 Gy with 6 MV linear accelerator was used to assess the efficacy of this proposed combined treatment. A triple negative breast cancer cell line, MDA-MB-231 was chosen as the model cell line. Monte Carlo (MC) calculations were done to predict the GNP-mediated cell death using the experimental GNP uptake data. Results: There was a 1.5- and 2- fold increase in uptake of 17 and 46 nm GNPs in the synchronized cell population, respectively. A radiation dose of 2 Gy with clinically relevant 6 MV photons resulted in a 62 and 38 % enhancement in cell death in the synchronized cell population with the incorporation of 17 and 46 nm GNPs, respectively. MC data supported the experimental data, but to a lesser extent. Conclusion: A triple combination of GNPs, cell cycle synchronization, and RT could pave the way to enhance the local radiation dose while minimizing side effects to the surrounding healthy tissue. Advances in knowledge: This is the first study to show that the combined use of GNPs, phase of tumor cell population, and RT could enhance tumor cell death.

Background: Radiotherapy is commonly used for treating cancer. Novel sensitizers, such as gold nanoparticles (GNPs), are being used to enhance the local radiation dose. It is not known how the uptake and radiation dose enhancement of GNPs vary in synchronized vs unsynchronized (control) tumor cell populations. Successful application of GNPs in radiation therapy requires NPs to be accumulated within individual tumor cells at clinically feasible NP concentrations. Use of small GNPs as a radiation dose enhancer in the past required very high NP concentration, since the driving force for the uptake of smaller GNPs is low. We used a novel lipid-based NP of 50 nm diameter system as a Trojan horse to deliver smaller GNPs of size 5 nm (LNP-GNP) at 0.2 nM concentration. We investigated the changes in GNP uptake and survival fraction with the LNP delivery at different cell stages using human breast cancer as our tumor model and choosing the triple-negative MDA-MB-231 cell line. Results: Using the LNP-GNP system resulted in a 39-and 73-fold enhancement in uptake of 5 nm GNPs in unsynchronized and synchronized tumor cell populations, respectively. The NP uptake per cell increased from 800 to 1200 and from 30,841 to 88,477 for individual 5 nm GNPs and 5 nm GNPs incorporated in LNPs, respectively. After a radiation dose of 2 Gy with 6 MeV photons, synchronized tumor cell populations incorporated with LNP-GNPs produced a 27% enhancement in tumor cell death compared to the control (unsynchronized; no GNPs; 2 Gy). The findings of our experimental results were supported by modeling predictions based on Monte Carlo calculations. Conclusions: This study clearly shows that the cell cycle, GNPs, and radiation therapy can be combined to improve outcome of cancer therapy. Using the experimental data, we estimated the predicted improvement for a clinical treatment plan where 30 fractions of 2 Gy radiation dose were given over a period of time. Enhanced uptake and radiation sensitivity of a synchronous tumor cell population would produce a significant improvement in cell killing. For example, synchronizing cells and the addition of LNP-GNPs into tumor cells produced a 1000-fold enhancement in cell killing. Because the agents used for cell synchronization are in clinical practice, this approach may be a

Infrapopliteal Percutaneous Transluminal Angioplasty Versus Bypass Surgery as First-Line Strategies in Critical Leg Ischemia: A Propensity Score Analysis Soderstrom MI, Arvela EM, Korhonen M et al. Ann Surg 2010;252:765-73.Conclusion: Infrapopliteal percutaneous angioplasty, when feasible, can be an effective first-line strategy achieving similar long-term results as bypass surgery in patients with critical limb ischemia (CLI) when redo procedures are actively used.Summary: Retrospective studies of patient characteristics in endovascular and surgical trials often differ significantly. Propensity score analysis can be used to adjust for important differences between treatment groups. In this study, propensity score analysis was used to compare outcomes in patients who underwent percutaneous transluminal angioplasty (PTA) or bypass surgery of infrapopliteal arteries. The study cohort consisted of 1023 nonrandomized patients treated for CLI, with 262 endovascular and 761 surgical revascularization procedures performed on tibial or pedal arteries. A propensity score was used for stratification and for adjustment in multivariable analysis. Choice of treatment was based on physician preference and treatment feasibility. PTA and bypass surgery achieved similar 5-year limb salvage (75.3% vs 76.0%), similar survival (47.5% vs 43.3%), and amputationfree survival (37.7% vs 32.7%). Rates of freedom from further revascularization were also similar (73.3% vs 74.4%). Freedom from surgical revascularization was higher after bypass surgery (94.3% vs 86.2%, P Ͻ .001). Outcomes were similar with propensity-score matched pairs except for freedom from surgical revascularization, which was higher in the bypass surgery group (91.4% vs 85.3% at 5 years, P ϭ .045). In patients who underwent isolated infrapopliteal revascularization, PTA was associated with better limb salvage (75.5% vs 68.0%, P ϭ .042). Differences in freedom from surgical revascularization were not significant (78.8% vs 85.2%, P ϭ .17). The difference in the limb salvage rate was not maintained in propensity score-matched pairs (P ϭ .12).Comment: Propensity score analysis is an interesting mathematical exercise. However, as pointed out by Dr J. F. Hamming in a discussion after the article, the authors are still comparing "apples with oranges," in that endovascular treatment was reserved for patients with shorter lesions and mostly stenosis and not occlusions. The underlying problem of trying to compare treatment modalities in patients with differing extent of disease still exists. What the paper really demonstrates is that it is possible to be reasonably good at individualizing treatment in patients with CLI. The overall effectiveness of the different treatment modalities in patients with equal burden of disease is not answered by this study.