Senescence induces miR-409 to down-regulate CCL5 and impairs angiogenesis in endothelial progenitor cells.

This study explores the impact of senescence on autocrine C-C motif chemokine ligand 5 (CCL5) in human endothelial progenitor cell (EPCs), addressing the poorly understood decline in number and function of EPCs during ageing. We examined the effects of replication-induced senescence on CCL5/CCL5 receptor (CCR5) signalling and angiogenic activity of EPCs in vitro and in vivo. We also explored microRNAs controlling CCL5 secretion in senescent EPCs, its impact on EPC angiogenic activity, and validated our findings in humans. CCL5 secretion and CCR5 levels in senescent EPCs were reduced, leading to attenuated angiogenic activity. CCL5 enhanced EPC proliferation via the CCR5/AKT/P70S6K axis and increased vascular endothelial growth factor (VEGF) secretion. Up-regulation of miR-409 in senescent EPCs resulted in decreased CCL5 secretion, inhibiting the angiogenic activity, though these negative effects were counteracted by the addition of CCL5 and VEGF. In a mouse hind limb ischemia model, CCL5 improved the angiogenic activity of senescent EPCs. Analysis involving 62 healthy donors revealed a negative association between CCL5 levels, age and Framingham Risk Score. These findings propose CCL5 as a potential biomarker for detection of EPC senescence and cardiovascular risk assessment, suggesting its therapeutic potential for age-related cardiovascular disorders.

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis.

Senescence reduces the circulating number and angiogenic activity of endothelial progenitor cells (EPCs), and is associated with aging-related vascular diseases. However, it is very time-consuming to obtain aged cells (~1 month of repeated replication) or animals (~2 years) for senescence studies. Here, we established an accelerated senescence model by treating EPCs with deferoxamine (DFO), an FDA-approved iron chelator. Four days of low-dose (3 µM) DFO induced senescent phenotypes in EPCs, including a senescent pattern of protein expression, impaired mitochondrial bioenergetics, altered mitochondrial protein levels and compromised angiogenic activity. DFO-treated early EPCs from young and old donors (< 35 vs. > 70 years old) displayed similar senescent phenotypes, including elevated senescence-associated ß-galactosidase activity and reduced relative telomere lengths, colony-forming units and adenosine triphosphate levels. To validate this accelerated senescence model in vivo, we intraperitoneally injected Sprague-Dawley rats with DFO for 4 weeks. Early EPCs from DFO-treated rats displayed profoundly senescent phenotypes compared to those from control rats. Additionally, in hind-limb ischemic mice, DFO pretreatment compromised EPC angiogenesis by reducing both blood perfusion and capillary density. DFO thus accelerates EPC senescence and appears to hasten model development for cellular senescence studies.

Ultrasonic microbubble VEGF gene delivery improves angiogenesis of senescent endothelial progenitor cells.

The therapeutic effects of ultrasonic microbubble transfection (UMT)-based vascular endothelial growth factor 165 (VEGF165) gene delivery on young and senescent endothelial progenitor cells (EPCs) were investigated. By UMT, plasmid DNA (pDNA) can be delivered into both young EPCs and senescent EPCs. In the UMT groups, higher pDNA-derived protein expression was found in senescent EPCs than in young EPCs. Consistent with this finding, a higher intracellular level of pDNA copy number was detected in senescent EPCs, with a peak at the 2-h time point post UMT. Ultrasonic microbubble delivery with or without VEGF improved the angiogenic properties, including the proliferation and/or migration activities, of senescent EPCs. Supernatants from young and senescent EPCs subjected to UMT-mediated VEGF transfection enhanced the proliferation and migration of human aortic endothelial cells (HAECs), and the supernatant of senescent EPCs enhanced proliferation more strongly than the supernatant from young EPCs. In the UMT groups, the stronger enhancing effect of the supernatant from senescent cells on HAEC proliferation was consistent with the higher intracellular VEGF pDNA copy number and level of protein production per cell in the supernatant from senescent cells in comparison to the supernatant from young EPCs. Given that limitations for cell therapies are the inadequate number of transplanted cells and/or insufficient cell angiogenesis, these findings provide a foundation for enhancing the therapeutic angiogenic effect of cell therapy with senescent EPCs in ischaemic cardiovascular diseases.

Enhanced Proliferation of Endothelial Progenitor Cells Post-Ultrasonic Microbubble Transfection Is Plasmid DNA Size Dependent and Contributed by Interleukin-6 Generation.

We investigated whether ultrasonic microbubble transfection (UMT) would enhance the transfection of large-sized luciferase plasmids (5.6, 9.2 and 33 kb) and biological impacts. Porcine venous blood endothelial progenitor cells (EPCs) were cultured in a medium containing plasmid DNA (pDNA) of different sizes followed by UMT and functional assays. Real-time polymerase chain reaction was conducted to investigate the effects of transfection of pDNA on multiple molecules central to endothelial function. The results indicated enhanced luciferase expression after UMT but the enhancement declined with increase in the size of the plasmid. UMT of pDNAs sized 5.6 and 9.2 kb into EPCs led to significant enhancement of proliferation. The interleukin-6 (IL-6) secreted from UMT of EPCs also increased in the 5.6- and 9.2-kb pDNA groups. Treatment of the transfected EPCs with anti-IL-6 antibody neutralized the proliferation. In conclusion, UMT of pDNAs sized 5.6 and 9.2 kb into EPCs increased the secretion of IL-6, which in turn enhanced cell proliferation.

Anti-angiogenic therapy renders large tumors vulnerable to immunotherapy via reducing immunosuppression in the tumor microenvironment.

We have recently demonstrated that a 4-in-1 gene therapy strategy that contains two anti-angiogenic genes [endostatin and pigment epithelium-derived factor] and two cytokine genes [granulocyte macrophage colony-stimulating factor and interleukin 12] has a considerable antitumor effect on large tumors in a woodchuck hepatoma model. The current study further investigates the underlying mechanisms for the antitumor effect observed by using small rodent models. We found that immunotherapy alone increased immunosuppressive cells in large tumors over time, whereas the anti-angiogenic therapy contained in the 4-in-1 strategy alleviated immunosuppression and made tumors vulnerable to immunotherapy, thus resulting in a synergistic antitumor effect.

Calreticulin promotes tumor lymphocyte infiltration and enhances the antitumor effects of immunotherapy by up-regulating the endothelial expression of adhesion molecules.

Tumor-induced angiogenesis has been shown to suppress immune responses. One mechanism is to suppress leukocyte-endothelial cell interaction by down-regulating the expression of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin on the tumor endothelium, which enables tumor cells to escape immune surveillance. Calreticulin (CRT), a chaperone protein mainly located in the endoplasmic reticulum, has been shown to exert anti-angiogenic activity and inhibit tumor growth. Here, we demonstrate that in addition to inhibiting angiogenesis, CRT also enhances the expression of both ICAM-1 and VCAM-1 on tumor endothelial cells. This expression results in enhanced leukocyte-endothelial cell interactions and increased lymphocyte infiltration into tumors. Therefore, combining intramuscular CRT gene transfer with intratumoral cytokine gene therapies significantly improves the antitumor effects of immunotherapy by markedly increasing the levels of tumor-infiltrating lymphocytes. This combined treatment increased the levels of infiltrating lymphocytes to those achieved using four times the cytokine dosage. The combined therapy also resulted in lower levels of immunosuppressive molecules and higher levels of activated T-cells in the tumor microenvironment than immunotherapy alone. In conclusion, this study describes a new antitumor mechanism of CRT that involves the up-regulation of tumor endothelial adhesion molecules and the enhanced infiltration of tumor-specific lymphocytes. Thus, CRT treatment can make tumor cells more vulnerable to immunotherapy and improve the therapeutic efficacy of immunotherapy.

Combining antiangiogenic therapy with immunotherapy exerts better therapeutical effects on large tumors in a woodchuck hepatoma model.

Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3(+) T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.