A dynamic magnetic shift method to increase nanoparticle concentration in cancer metastases: a feasibility study using simulations on autopsy specimens

A nanoparticle delivery system termed dynamic magnetic shift (DMS) has the potential to more effectively treat metastatic cancer by equilibrating therapeutic magnetic nanoparticles throughout tumors. To evaluate the feasibility of DMS, histological liver sections from autopsy cases of women who died from breast neoplasms were studied to measure vessel number, size, and spatial distribution in both metastatic tumors and normal tissue. Consistent with prior studies, normal tissue had a higher vascular density with a vessel-to-nuclei ratio of 0.48 ± 0.14 (n = 1000), whereas tumor tissue had a ratio of 0.13 ± 0.07 (n = 1000). For tumors, distances from cells to their nearest blood vessel were larger (average 43.8 μm, maximum 287 μm, n ≈ 5500) than normal cells (average 5.3 μm, maximum 67.8 μm, n ≈ 5500), implying that systemically delivered nanoparticles diffusing from vessels into surrounding tissue would preferentially dose healthy instead of cancerous cells. Numerical simulations of magnetically driven particle transport based on the autopsy data indicate that DMS would correct the problem by increasing nanoparticle levels in hypovascular regions of metastases to that of normal tissue, elevating the time-averaged concentration delivered to the tumor for magnetic actuation versus diffusion alone by 1.86-fold, and increasing the maximum concentration over time by 1.89-fold. Thus, DMS may prove useful in facilitating therapeutic nanoparticles to reach poorly vascularized regions of metastatic tumors that are not accessed by diffusion alone

Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole

Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC50 dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy

Cyclosporin a disrupts notch signaling and vascular lumen maintenance.

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos

Notch inhibition partially rescues CSA induced vascular malfunction.

<p>(A) Effect of CSA and DAPT on vascular function in zebrafish embryos. Freshly laid Fli1-GFP / GATA1-RFP zebrafish embryos were incubated in 0.1% DMSO (Control), 10μM CSA, 15μM DAPT, or 10μM CSA + 15μM DAPT for 2 days. Bright field imaging revealed no gross morphological abnormalities in either CSA or DAPT treated fish, however CSA + DAPT treated fish experienced an acute curvature. GFP imaging revealed a lack of lumen structures in the ISV (white arrowheads) and aortic vessels (red arrowheads) of CSA treated fish. DAPT treated fish displayed normal luminal structure and blood flow. CSA + DAPT treated embryos had luminal structures (arrowheads) and blood flow similar to control or DAPT alone treated embryos. Shown are representative results from a single experiment that was performed five times in its entirety. (B) Quantitative analysis of blood flow in zebrafish treated with CSA, DAPT, or CSA + DAPT. Data shown represent the average +/− SE of five individual experiments. P-values were determined by student’s t-test.</p

CSA blocks Notch signaling.

<p>(A) Effect of CSA on Notch signaling <i>in vitro</i>. 293T cells were transfected with various combinations of myc-tagged murine Notch1 (N), JAG1 (J), or Delta-like 4 (D) and treated with either 0.1% DMSO or 10μM CSA. Whole cell lysates were fractionated through SDS-PAGE gels and western blotted with anti-Val1744 antibody to detect cleaved Notch1 NICD fragments (N1ICD). Stripped blots were re-blotted with β-actin or 9E10 anti-myc antibodies to control for protein loading and expression of various transfected cDNAs. Shown are representative western blots from a single experiment that was performed five times in its entirety. (B) Western blot quantitation comparing N1ICD levels in cells transfected with Notch1 alone to cells transfected with combinations of Notch and JAG1 or Dll4 in the presence or absence of CSA. Displayed data represent the mean +/− SE of five individual experiments. P-values were calculated with the Student’s t-test. (C) Effects of CSA on Notch activity <i>in</i> vivo. Tp1bglob:eGFP embryos which express GFP from a tandem array of 12 Notch responsive RBP-Jk binding sites were incubated in either 0.1% DMSO, 10μM DAPT, or 10μM CSA. 48 hours later, GFP signal intensity was quantified in whole, live embryos. Data shown represents the mean +/− SE of 4 individual experiments. P-values were determined by student’s t-test. (D) Representative pictures of Tp1bglob:eGFP zebrafish embryos incubated with 10M DAPT or 10M CSA and imaged by fluorescent microscopy.</p

Cyclosporin-A destabilizes vascular lumen structures in zebrafish embryos.

<p>Freshly laid Fli1-GFP/GATA1-RFP zebrafish embryos were incubated in 10μM CSA or DMSO vehicle control for one, two, or four days. Whole embryo brightfield imaging was used to monitored gross morphology. Development of the vascular system was monitored by fluorescent microscopy of endothelial specific GFP expression. Circulatory flow was monitored by fluorescent microscopy of red-blood cell specific RFP expression. (A) Effects of CSA on 1dpf embryos. 1 day after CSA treatment, brightfield imaging of zebrafish embryos (top panel) was unable to distinguish any significant developmental impact of CSA on gross embryo morphology. Microangiogram analysis revealed similar development of the primitive vascular system including sprouting intersegmental vessels. (B) Effects of CSA on 2 dpf embryos. Zebrafish embryos treated with CSA for two days displayed no obvious signs of developmental abnormality in bright field images. Low power GFP imaging revealed an apparently normal vascular system, however RFP imaging revealed a distinct lack of blood flow throughout the embryo. High power GFP imaging revealed a lack of vascular lumen structures in ISV structures (arrows). (C) Effect of CSA on 4 dpf embryos. After four days of CSA treatment, no vascular luminal structures (arrows) or blood flow was evident in CSA treated embryos.</p

Inhibition of cyclophilin A or calcineurin/NFAT destabilizes lumen structure.

<p>(A) Freshly laid Fli1-GFP / GATA1-RFP embryos were incubated in 0.1% DMSO, 2μM FK506, or 40μM <i>N</i>-MeVal-4-CsA (CSA-Analog) for two days. Fluorescent imaging was used to monitor overall vascular development (GFP) and blood flow (RFP). Similar to CSA, neither FK506 nor <i>N</i>-MeVal-4-CSA had an impact on gross morphology or overall vascular development however both drugs blocked blood flow.</p

Traumatic ventricular septal defect resulting in severe pulmonary hypertension.

Traumatic ventricular septal defect (VSD) is a widely-recognized complication of both penetrating and blunt trauma. Most cases are repaired operatively without the long-term complications of pulmonary hypertension and heart failure that are associated with unrepaired congenital VSD in the pediatric population. To our knowledge, this is the first case report of a patient with a traumatic VSD who declined surgical repair at the time of injury and subsequently developed long-term complications of pulmonary hypertension and heart failure. With nearly 20 years of follow-up, this case demonstrates that the absence of surgical treatment in asymptomatic adult patients at the time of injury can lead to long-term complications associated with VSD. This case also shows that aggressive surgical treatment in patients with severe pulmonary vascular disease and heart failure secondary to traumatic VSD can be performed safely and should be considered in cases refractory to efficacious medical interventions