Effect of Ambient Conditions on Acoustic Activation of the Perfluoropropane Droplets Within the Infarct Zone.

BACKGROUND: Acoustically activated perfluoropropane droplets (PD) formulated from lipid encapsulated microbubble preparations produce a delayed myocardial contrast enhancement that preferentially highlights the infarct zones (IZ). Since activation of PDs may be temperature sensitive, it is unclear what effect body temperature (BT) has on acoustic activation (AA). OBJECTIVE: We sought to determine whether the microvascular retention and degree of myocardial contrast intensity (MCI) would be affected by BT at the time of intravenous injection. METHODS: We administered intravenous (IV) PD in nine rats following 60 min of ischemia followed by reperfusion. Injections in these rats were given at temperatures above and below 36.5°C, with high MI activation in both groups at 3 or 6 min following IV injection (IVI). In six additional rats (three in each group), IV PDs were given only at one temperature (<36.5°C or ≥36.5°C), permitting a total of 12 comparisons of different BT. Differences in background subtracted MCI at 3-6 min post-injection were compared in the infarct zone (IZ) and remote zone (RZ). Post-mortem lung hematoxylin and eosin (H&E) staining was performed to assess the effect potential thermal activation on lung tissue. RESULTS: Selective MCI within the IZ was observed in 8 of 12 rats who received IVI of PDs at <36.5°C, but none of the 12 rats who had IVI at the higher temperature (p < 0.0001). Absolute MCI following droplet activation was significantly higher in both the IZ and RZ when given at the lower BT. H&E indicated significant red blood extravasation in 5/7 rats who had had IV injections at higher BT, and 0/7 rats who had IV PDs at <36.5°C. CONCLUSIONS: Selective IZ enhancement with AA of intravenous PDs is possible, but temperature sensitive. Thermal activation appears to occur when PDs are given at higher temperatures, preventing AA, and increasing unwanted bioeffects.

Acoustic Activation Imaging With Intravenous Perfluoropropane Nanodroplets Results in Selective Bioactivation of the Risk Area.

BACKGROUND: Acoustically activatable perfluoropropane droplets (PD) can be formulated from commercially available microbubble preparations. Diagnostic transthoracic ultrasound frequencies have resulted in acoustic activation (AA) predominately within myocardial infarct zones (IZ). OBJECTIVE: We hypothesized that the AA area following acute coronary ischemia/reperfusion (I/R) would selectively enhance the developing scar zone, and target bioeffects specifically to this region. METHODS: We administered intravenous PD in 36 rats and 20 pigs at various stages of myocardial scar formation (30 minutes, 1 day, and 7 days post I/R) to determine what effect infarct age had on the AA within the IZ. This was correlated with histology, myeloperoxidase activity, and tissue nitrite activity. RESULTS: The degree of AA within the IZ in rats was not associated with collagen content, neutrophil infiltration, or infarct age. AA within 24 hours of I/R was associated with increased nitric oxide utilization selectively within the IZ (P < .05 compared with remote zone). The spatial extent of AA in pigs correlated with infarct size only when performed before sacrifice at 7 days (r = .74, P < .01). CONCLUSIONS: Acoustic activation of intravenous PD enhances the developing scar zone following I/R, and results in selective tissue nitric oxide utilization.

Targeted deletion of p53 in the proximal tubule prevents ischemic renal injury.

The contribution of p53 to kidney dysfunction, inflammation, and tubular cell death, hallmark features of ischemic renal injury (IRI), remains undefined. Here, we studied the role of proximal tubule cell (PTC)-specific p53 activation on the short- and long-term consequences of renal ischemia/reperfusion injury in mice. After IRI, mice with PTC-specific deletion of p53 (p53 knockout [KO]) had diminished whole-kidney expression levels of p53 and its target genes, improved renal function, which was shown by decreased plasma levels of creatinine and BUN, and attenuated renal histologic damage, oxidative stress, and infiltration of neutrophils and macrophages compared with wild-type mice. Notably, necrotic cell death was attenuated in p53 KO ischemic kidneys as well as oxidant-injured p53-deficient primary PTCs and pifithrin-α-treated PTC lines. Reduced oxidative stress and diminished expression of PARP1 and Bax in p53 KO ischemic kidneys may account for the decreased necrosis. Apoptosis and expression of proapoptotic p53 targets, including Bid and Siva, were also significantly reduced, and cell cycle arrest at the G2/M phase was attenuated in p53 KO ischemic kidneys. Furthermore, IRI-induced activation of TGF-ß and the long-term development of inflammation and interstitial fibrosis were significantly reduced in p53 KO mice. In conclusion, specific deletion of p53 in the PTC protects kidneys from functional and histologic deterioration after IRI by decreasing necrosis, apoptosis, and inflammation and modulates the long-term sequelae of IRI by preventing interstitial fibrogenesis.

Tumor- and organ-dependent infiltration by myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) increase during tumor growth and following cytoreductive therapy resulting in immune dysfunction and tumor escape from host control. We report organ- and tumor-specific expansion of MDSCs, differences in their molecular and membrane phenotypes and T-cell suppressive activity. A significant increase in MDSCs was observed within the spleen, peripheral blood (PB), bone marrow (BM), lungs, and livers of mice bearing orthotopic 4T1, but not CI66 mammary tumors. The PB of 4T1 TB mice had the highest frequency of MDSCs (78.6±2.1%). Similarly, the non-parenchymal cells (NPCs) in the tumor tissue, livers and lungs of 4T1 tumor-bearing (TB) mice had an increased MDSCs frequency. Studies into Gr-1 and Ly-6C staining of MDSCs revealed significant increases in CD11b+Gr-1(dull)Ly-6C(high) and CD11b+Gr-1(bright)Ly-6C(low) subsets. The frequency of MDSCs inversely correlated with the CD3+ T-cell frequency in the spleen, and blood of 4T1 TB mice and was associated with a significant decrease in splenic and NPCs IFN-γ and IL-12 transcript levels, as well as significantly increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-10 (IL-10), interleukin-13 (IL-13), arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor-A (VEGF-A) transcripts. In summary, MDSCs are significantly increased not only in lymphoid organs, but also in parenchymal organs including lungs and livers of TB mice, where they may facilitate metastasis to these organ sites.

Therapeutic activity of sunitinib for Her2/neu induced mammary cancer in FVB mice.

Mouse mammary tumor virus-Neu (MMTV/neu) transgenic mice on an FVB-background (FVB-neuN) have increased numbers of myeloid derived suppressor cells (MDSCs) and regulatory T-cells (T-regs) in the spleen during mammary tumor induction and progression. Using this transgenic tumor model, we assessed the therapeutic activity of sunitinib, a multi-targeted, tyrosine kinase (TK) inhibitor and its effects on immune-regulatory cells. Our preliminary results show that sunitinib at 40mg/kg/day, p.o. (per os), delayed the time to tumor induction and reduced the incidence and growth of tumors in FVB-neuN mice. In association with its therapeutic activity, sunitinib reduced the absolute number of splenic T-reg cells (CD4(+)CD25(+)CD62L(+)) and MDSCs (CD11b(+)Gr1(+)) that were increased during tumor progression with less activity in mice with gross tumors. A significant decrease in the absolute number of splenic T-regs, dendritic cells (DCs), MDSCs and hematopoietic progenitors (Lin(-)Sca1(+)CD90(dull)) was observed following sunitinib treatment. The frequency of splenic T-regs and hematopoietic progenitors, but not MDSCs was also reduced by sunitinib treatment. Additionally immune-regulatory cytokines and enzymes were down regulated by sunitinib treatment, including TGFbeta and NOS2 in the spleen cells of sunitinib treated mice as compared to untreated tumor bearing (TB) mice. We conclude that sunitinib has therapeutic activity, in association with the down regulation of MDSCs and T-regs and has a trend towards the normalization of the inflammatory cytokine levels induced by tumor progression and growth. Based on these results, we suggest that sunitinib reduction of immune suppressive cells is a critical part of its adjuvant immune therapeutic activity.

Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice.

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1(+)CD11b(+) myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147-579) and 329 (161-523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4(+) T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-gamma, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.