A Novel Dextran-Based Dual Drug Conjugate Targeted Tumors with High Biodistribution Ratio of Tumors to Normal Tissues.

Purpose: Most chemotherapeutic agents possess poor water solubility and show more significant accumulations in normal tissues than in tumor tissues, resulting in serious side effects. To this end, a novel dextran-based dual drug delivery system with high biodistribution ratio of tumors to normal tissues was developed. Methods: A bi-functionalized dextran was developed, and several negatively charged dextran-based dual conjugates containing two different types of drugs, docetaxel and docosahexaenoic acid (DTX and DHA, respectively) were synthesized. The structures of these conjugates were characterized using nuclear magnetic resonance and liquid chromatography/mass spectrometry (1H-NMR and LC/MS, respectively) analysis. Cell growth inhibition, apoptosis, cell cycle distribution, and cellular uptake were measured in vitro. Drug biodistribution and pharmacokinetics were investigated in mice bearing 4T1 tumors using LC/MS analysis. Drug biodistribution was also explored by in vivo imaging. The effects of these conjugates on tumor growth were evaluated in three mice models. Results: The dextran-docosahexaenoic acid (DHA)- docetaxel (DTX) conjugates caused a significant enhancement of DTX water solubility and improvement in pharmacokinetic characteristics. The optimized dextran-DHA-DTX conjugate A treatment produced a 2.1- to 15.5-fold increase in intra-tumoral DTX amounts for up to 96 h compared to parent DTX treatment. Meanwhile, the concentrations of DTX released from conjugate A in normal tissues were much lower than those of the parent DTX. This study demonstrated that DHA could lead to an improvement in the efficacy of the conjugates and that the conjugate with the shortest linker displayed more activity than conjugates with longer linkers. Moreover, conjugate A completely eradicated all MCF-7 xenograft tumors without causing any obvious side effects and totally outperformed both the conventional DTX formulation and Abraxane in mice. Conclusion: These dextran-based dual drug conjugates may represent an innovative tumor targeting drug delivery system that can selectively deliver anticancer agents to tumors.

Complete regression of xenografted breast tumors by dextran-based dual drug conjugates containing paclitaxel and docosahexaenoic acid.

In this study, a novel carboxymethyl dextran (CMD)-based dual drug delivery system that delivering two water insoluble drugs to tumor sites was developed and evaluated for anticancer activities. Paclitaxel (PTX) and docosahexaenoic acid (DHA) were covalently coupled with CMD to generate CMD-DHA-PTX conjugate S and conjugate L with different linkers containing amino acids Gly-Gly or Lys-Gly-Gly, respectively. Both conjugates possessed high PTX loading contents and enhanced water solubility, as well as the ability of being self-assembled into nanoparticles with the nanoparticle size ranged from 88.7 nm to 94.7 nm. These two conjugates released free PTX continuously in plasma and cancer cells. The conjugate S exhibited improved pharmacokinetic parameters and higher distribution extent in tumor sites than the parent PTX, Abraxane and the conjugate L. The antitumor efficacy of these two conjugates outperformed parent PTX formulation and Abraxane in nude mice bearing breast cancer cells MCF-7. More importantly, the conjugate S treatment eliminated all the xenograft tumors without causing any mice body weight loss in mice model. This study revealed that the dextran-based dual drug conjugates may represent an effective and innovative way to deliver anticancer agents to a variety of tumors.

Percutaneous Coronary Intervention Through a Retrograde Approach in Complete Occlusion of the Coronary Ostium Following Surgical Aortic Valve Replacement.

Coronary ostial stenosis, treated by either percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), is a rare but potentially life-threatening complication following surgical aortic valve replacement. However, in cases of complete occlusion of the coronary ostium (COCO), CABG is the typical treatment because guiding catheter engagement is extremely challenging. Herein, we presented a case of a patient with COCO who underwent successful PCI through a retrograde approach, and this case first demonstrates the feasibility of PCI for the treatment of COCO, which could help avoid the high risk of redo surgery.

Attenuation of lung cancer stem cell tumorigenesis and metastasis by cisplatin.

PURPOSE: To investigate the effect of cisplatin on the growth and metastasis abilities of lung cancer stem cells (CSCs) via molecular imaging. MATERIALS AND METHODS: The expression changes of lung CSCs cell marker in A549-Luc-C8 human non-small-cell lung cancer (NSCLC) cell line with or without cisplatin treatment were detected by flow cytometry. The tumorigenesis and metastasis abilities of A549-Luc-C8 cells were monitored both in vitro and in vivo, and the mechanism was assessed by gene sequencing. RESULTS: About 1%-2% of CSCs were detected in A549-Luc-C8 cells and decreased CSCs percentage was observed after cisplatin treatment. Attenuated tumorigenesis and metastasis abilities of A549-Luc-C8 cells were found in cisplatin treated group. CONCLUSIONS: Decreased percentage of CSCs in A549-Luc-C8 cells can be induced by cisplatin treatment, which may partly be attributed to the attenuated expression of growth factors.

Noninvasive visualization of microRNA-16 in the chemoresistance of gastric cancer using a dual reporter gene imaging system.

MicroRNAs (miRNAs) have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of (131)I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo (99m)Tc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16) and 5-fluorouracil (5-FU) activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic.

Molecular imaging of p53 signal pathway in lung cancer cell cycle arrest induced by cisplatin.

Cisplatin is a commonly employed chemotherapy drug for lung malignancy. However its efficacy is limited by acquired drug resistance and lacking of an in vivo real-time monitoring approach. The aim of this study is to investigate the effect of cisplatin on lung adenocarcinoma cell line p53-RE-Fluc/A549 in vivo via non-invasive reporter gene by molecular imaging. For this study, we employed p53-RE-Fluc/A549 cells that overexpressed a vector with three tandem repeats of p53 response element followed by the luciferase reporter gene. P53 activity was evaluated by optical imaging and verified by Western blot after cells were exposed to 10 µM cisplatin for 72 h. The cell cycle was mainly blocked at the S- and G2/M-phases after cisplatin treatment, whereas no significant change was observed in cell apoptotic index. Increased expression of p21 and Bcl-2 as well as decreased expression of Bax were observed after cisplatin treatment by Western blotting. Longitudinal in vivo bioluminescent imaging (BLI) revealed that the p53 activity was increased from 24 to 48 h after transient cisplatin treatment in p53-RE-Fluc/A549-bearing nude mice. RNA sequencing further revealed that cell cycle and p53 signaling pathway genes, such as E2F1, CCNA2, CDK1, and CCNE2 were significantly downregulated after long-term cisplatin treatment. Thus, our study showed that cisplatin exerts its cytotoxic effect through blockage of the cell cycle and may be partly regulated by the p53 signaling pathway. Furthermore, molecular imaging is a useful tool to investigate the mechanism and evaluate the effect of chemotherapy drugs both in vivo and in vitro.

mTORC1 and mTORC2 play different roles in the functional survival of transplanted adipose-derived stromal cells in hind limb ischemic mice via regulating inflammation in vivo.

Poor cell survival severely limits the beneficial effects of stem cell therapy for peripheral arterial disease (PAD). This study was designed to investigate the role of mammalian target of rapamycin (mTOR) in the survival and therapeutic function of transplanted murine adipose-derived stromal cells (mADSCs) in a murine PAD model. mADSCs (1.0 × 10(7)) were isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein-positive transgenic mice, intramuscularly implanted into the hind limb of C57BL/6 mice after femoral artery ligation/excision, and monitored using noninvasive bioluminescence imaging (BLI). Although engrafted mADSCs produced antiapoptotic/proangiogenic effects in vivo by modulating the inflammatory and angiogenic cytokine response involving the mTOR pathway, longitudinal BLI revealed progressive death of post-transplant mADSCs within ~4 weeks in the ischemic hind limb. Selectively targeting mTOR complex-1 (mTORC1) using low-dose rapamycin treatment with mADSCs attenuated proinflammatory cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]) expression and neutrophil/macrophage infiltration, which overtly promoted mADSCs viability and antiapoptotic/proangiogenic efficacy in vivo. However, targeting dual mTORC1/mTORC2 using PP242 or high-dose rapamycin caused IL-1ß/TNF-α upregulation and anti-inflammatory IL-10, IL-6, and vascular endothelial growth factor/vascular endothelial growth factor receptor 2 downregulation, undermining the survival and antiapoptotic/proangiogenic action of mADSCs in vivo. Furthermore, low-dose rapamycin abrogated TNF-α secretion by mADSCs and rescued the cells from hypoxia/reoxygenation-induced death in vitro, while PP242 or high-dose rapamycin exerted proinflammatory effects and promoted cell death. In conclusion, mTORC1 and mTORC2 may differentially regulate inflammation and affect transplanted mADSCs' functional survival in ischemic hind limb. These findings uncover that mTOR may evolve into a promising candidate for mechanism-driven approaches to facilitate the translation of cell-based PAD therapy.

Adipose stromal cell and sarpogrelate orchestrate the recovery of inflammation-induced angiogenesis in aged hindlimb ischemic mice.

Aging population displays a much higher risk of peripheral arterial disease (PAD) possibly due to the higher susceptibility, poor prognosis, and fewer therapeutic options. This study was designed to examine the impact of combined multipotent adipose-derived stromal cells (mADSCs) and sarpogrelate treatment on aging hindlimb ischemia and the mechanism of action involved. mADSCs (1.0 × 10(7)) constitutively expressing enhanced green fluorescent protein (eGFP) or firefly luciferase (Fluc) reporter were engrafted into the hindlimb of aged Vegfr2-luc transgenic or FVB/N mice subjected to unilateral femoral artery occlusion, followed by a further administration of sarpogrelate. Multimodality molecular imaging was employed to noninvasively evaluate mADSCs' survival and therapeutic efficacy against aging hindlimb ischemia. Aged Tg(Vegfr2-luc) mice exhibited decreased inflammatory response, and downregulation of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) compared with young ones following hindlimb ischemia induction, resulting in angiogenesis insufficiency and decompensation for ischemia recovery. Engrafted mADSCs augmented inflammation-induced angiogenesis to yield pro-angiogenic/anti-apoptotic effects partly via the VEGF/VEGFR2/mTOR/STAT3 pathway. Nonetheless, mADSCs displayed limited survival and efficacy following transplantation. Sarpogrelate treatment with mADSCs further upregulated mammalian target of rapamycin (mTOR)/STAT3 signal and modulated pro-/anti-inflammatory markers including IL-1ß/TNF-α/IFN-γ and IL-6/IL-10, which ultimately facilitated mADSCs' survival and therapeutic benefit in vivo. Sarpogrelate prevented mADSCs from hypoxia/reoxygenation-induced cell death via a mTOR/STAT3-dependent pathway in vitro. This study demonstrated a role of in vivo kinetics of VEGFR2 as a biomarker to evaluate cell-derived therapeutic angiogenesis in aging. mADSCs and sarpogrelate synergistically restored impaired angiogenesis and inflammation modulatory capacity in aged hindlimb ischemic mice, indicating its therapeutic promise for PAD in the elderly.

Effects of hepatocyte growth factor overexpressed bone marrow-derived mesenchymal stem cells on prevention from left ventricular remodelling and functional improvement in infarcted rat hearts.

Bone marrow-derived mesenchymal stem cells (BM-MSCs ) transplantation has been reported to be a promising therapy for myocardial infarction (MI). However, low survival rate of BM-MSCs in infarcted heart is one of the major limitations for the perspective clinical application. In this study, we aimed to investigate the effect of hepatocyte growth factor (HGF) on left ventricular function improvement of HGF gene-modified BM-MSCs (HGF-MSCs) after its delivery into the infarcted rat hearts. BM-MSCs were isolated with fibroblast-like morphology and expressed CD44+CD29+CD90+/CD34-CD45-CD31-CD11a. After 5-azacytidine induction in vitro, 20%-30% of the cells were positively stained for desmin, cardiac-specific cardiac troponin I and connexin-43. Histological staining revealed that 2 weeks after MI is an optimal time point with decreased neutrophil infiltration and increased vascular number. Minimal infarct size and best haemodynamic analysis were also observed after cell injection at 2 weeks compared with that of 1 h, 1 week or 4 weeks. Echocardiogram confirmed that transplantation with HGF-MSCs significantly improved left ventricular function compared with other groups in rat MI models. MSCs and HGF-MSCslabelled with DAPI were detected 4 weeks after MI in the infarcted area. Decreased infarcted scar area and increased angiogenesis formation could be found in HGF-MSCs group than in other groups as demonstrated by hematoxylin and eosin (H&E) staining and factor VIII staining. These results indicate that HGF-MSCs transplantation could enhance the contractile function and attenuate left ventricular remodelling efficiently in rats with MI.

The synergistic effect of valsartan and LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] on vascular oxidative stress and inflammation in type 2 diabetic mice.

AIM: To investigate the combination effects and mechanisms of valsartan (angiotensin II type 1 receptor blocker) and LAF237 (DPP-IV inhibitor) on prevention against oxidative stress and inflammation injury in db/db mice aorta. METHODS: Db/db mice (n = 40) were randomized to receive valsartan, LAF237, valsartan plus LAF237, or saline. Oxidative stress and inflammatory reaction in diabetic mice aorta were examined. RESULTS: Valsartan or LAF237 pretreatment significantly increased plasma GLP-1 expression, reduced apoptosis of endothelial cells isolated from diabetic mice aorta. The expression of NAD(P)H oxidase subunits also significantly decreased resulting in decreased superoxide production and ICAM-1 (fold change: valsartan : 7.5 ± 0.7, P < 0.05; LAF237: 10.2 ± 1.7, P < 0.05), VCAM-1 (fold change: valsartan : 5.2 ± 1.2, P < 0.05; LAF237: 4.8 ± 0.6, P < 0.05), and MCP-1 (fold change: valsartan: 3.2 ± 0.6, LAF237: 4.7 ± 0.8; P < 0.05) expression. Moreover, the combination treatment with valsartan and LAF237 resulted in a more significant increase of GLP-1 expression. The decrease of the vascular oxidative stress and inflammation reaction was also higher than monotherapy with valsartan or LAF237. CONCLUSION: These data indicated that combination treatment with LAF237 and valsartan acts in a synergistic manner on vascular oxidative stress and inflammation in type 2 diabetic mice.

Effects of ghrelin on homocysteine-induced dysfunction and inflammatory response in rat cardiac microvascular endothelial cells.

Ghrelin is a well-characterized hormone that has protective effects on endothelial cells. Elevated HCY (homocysteine) can be a cardiovascular risk factor, but it is not known whether ghrelin can inhibit HCY-induced dysfunction and inflammatory response in rat CMECs (cardiac microvascular endothelial cells). We found that HCY treatment for 24 h inhibited proliferation and NO (nitric oxide) secretion, but with increased cell apoptosis and secretion of cytokines in CMECs. In contrast, ghrelin pretreatment significantly improved proliferation and NO secretion, and inhibited cell apoptosis and secretion of cytokines in HCY-induced CMECs. In addition, Western blot assay showed that NF-κB (nuclear factor κB) and cleaved-caspase 3 expression were elevated, and PCNA (proliferating cell nuclear antigen) and eNOS (endothelial nitric oxide synthase) expression were decreased after treatment with HCY, which was significantly reversed by pretreatment with ghrelin. The data suggest that ghrelin inhibits HCY-induced CMEC dysfunction and inflammatory response, probably mediated by inhibition of NF-κB activation.