Dual stimuli-responsive mesoporous silica nanoparticles for efficient loading and smart delivery of doxorubicin to cancer with RGD-integrin targeting.

The recent progress in nanoparticle applications, such as tumor-targeting, has enabled specific delivery of chemotherapeutics to malignant tissues with enhanced local efficacy while limiting side effects. However, existing delivery systems leave much room for improvement in terms of achieving enhanced colloidal stability in fluid medium, efficient targeting of intended sites, and effective release of therapeutic drugs into diseased cells. Here, an efficient stimuli-responsive nanocarrier for mammalian cells, termed RGD-NAMs, was developed, which enabled temperature- and pH-sensitive release of drug loads. The RGD-NAMs comprise two parts: a stimuli-responsive copolymer shell (NIBIm-AA-RGD) and drug-container core (MSNs). The RGD-NAMs have a stable drug-loading capacity with a marked difference in the release rate depending on the temperature and pH conditions. The RGD-NAMs also exhibit high colloidal stability in SBF (Stimulated body fluid) solutions and minimal toxicity in skeletal myoblasts (C2C12) and bovine arterial endothelial cells (BAEC). The doxorubicin-loaded RGD-NAMs induced a cytotoxic effect in a dose-dependent manner, which was furthered by an increase in temperature from 37 to 40 °C. Moreover, significant control of the release rate and the amount were achieved through pH change. This novel, smart drug-delivery system with high responsiveness to temperature and pH changes has wide application prospects in biomedical fields, including the theragnosis of tumors and vascular diseases.

Recent Progress and Opportunities for Nucleic Acid Aptamers.

Coined three decades ago, the term aptamer and directed evolution have now reached their maturity. The concept that nucleic acid could modulate the activity of target protein as ligand emerged from basic science studies of viruses. Aptamers are short nucleic acid sequences capable of specific, high-affinity molecular binding, which allow for therapeutic and diagnostic applications. Compared to traditional antibodies, aptamers have several advantages, including small size, flexible structure, good biocompatibility, and low immunogenicity. In vitro selection method is used to isolate aptamers that are specific for a desired target from a randomized oligonucleotide library. The first aptamer drug, Macugen, was approved by FDA in 2004, which was accompanied by many studies and clinical investigations on various targets and diseases. Despite much promise, most aptamers have failed to meet the requisite safety and efficacy standards in human clinical trials. Amid these setbacks, the emergence of novel technologies and recent advances in aptamer and systematic evolution of ligands by exponential enrichment (SELEX) design are fueling hope in this field. The unique properties of aptamer are gaining renewed interest in an era of COVID-19. The binding performance of an aptamer and reproducibility are still the key issues in tackling current hurdles in clinical translation. A thorough analysis of the aptamer binding under varying conditions and the conformational dynamics is warranted. Here, the challenges and opportunities of aptamers are reviewed with recent progress.

Ultrasound contrast-enhanced study as an imaging biomarker for anti-cancer drug treatment: preliminary study with paclitaxel in a xenograft mouse tumor model (secondary publication).

PURPOSE: The purpose of this study was to assess tumor angiogenesis using contrast-enhanced ultrasonography (CEUS) of human prostate cancer cells (PC3) that were implanted in mice before and after paclitaxel injection. METHODS: Twelve mice were injected with human PC3. The mice were grouped into two groups; one was the paclitaxel-treated group (n=6) and the other was the control group (n=6). Before administering paclitaxel into the peritoneal cavity, baseline CEUS was performed after the administration of 500 µL (1×108 microbubbles) of contrast agent. The area under the curve (AUC) up to 50 seconds after injection was derived from the time-intensity curves. After injection of paclitaxel or saline, CEUS studies were performed at the 1-week follow-up. Changes in tumor volume and the AUC in both two groups were evaluated. After CEUS, the microvessel density (MVD) was compared between the groups. RESULTS: In the paclitaxel-treated group, the AUC from CEUS showed a significant decrease 1-week after paclitaxel administration (P=0.030), even though the tumor volume showed no significant changes (P=0.116). In the control group, there was no significant decrease of the AUC (P=0.173). Pathologically, there was a significant difference in MVD between both groups (P=0.002). CONCLUSION: The AUC from the time intensity curve derived from CEUS showed an early change in response to the anti-cancer drug treatment that preceded the change in tumor size. The findings of CEUS could serve as an imaging biomarker for assessing tumor responses to anti-cancer drug treatment.

Emerging frontiers of graphene in biomedicine.

Graphene is a next-generation biomaterial with increasing biomedical applicability. As a new class of one-atom-thick nanosheets, it is a true two-dimensional honeycomb network nanomaterial that attracts interest in various scientific fields and is rapidly becoming the most widely studied carbon-based material. Since its discovery in 2004, its unique optical, mechanical, electronic, thermal, and magnetic properties are the basis of exploration of the potential applicability of graphene. Graphene materials, such as graphene oxide and its reduced form, are studied extensively in the biotechnology arena owing to their multivalent functionalization and efficient surface loading with various biomolecules. This review provides a brief summary of the recent progress in graphene and graphene oxide biological research together with current findings to spark novel applications in biomedicine. Graphene-based applications are progressively developing; hence, the opportunities and challenges of this rapidly growing field are discussed together with the versatility of these multifaceted materials.

Screening and characterization of a novel RNA aptamer that specifically binds to human prostatic acid phosphatase and human prostate cancer cells.

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2'-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2'-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.

Emerging roles of human prostatic Acid phosphatase.

Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed.

Nucleic Acid aptamers: new methods for selection, stabilization, and application in biomedical science.

The adoption of oligonucleotide aptamer is well on the rise, serving an ever increasing demand for versatility in biomedical field. Through the SELEX (Systematic Evolution of Ligands by EXponential enrichment), aptamer that can bind to specific target with high affinity and specificity can be obtained. Aptamers are single-stranded nucleic acid molecules that can fold into complex threedimensional structures, forming binding pockets and clefts for the specific recognition and tight binding of any given molecular target. Recently, aptamers have attracted much attention because they not only have all of the advantages of antibodies, but also have unique merits such as thermal stability, ease of synthesis, reversibility, and little immunogenicity. The advent of novel technologies is revolutionizing aptamer applications. Aptamers can be easily modified by various chemical reactions to introduce functional groups and/or nucleotide extensions. They can also be conjugated to therapeutic molecules such as drugs, drug containing carriers, toxins, or photosensitizers. Here, we discuss new SELEX strategies and stabilization methods as well as applications in drug delivery and molecular imaging.

Noninvasive molecular biomarkers for the detection of colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancy in the world. Because CRC develops slowly from removable precancerous lesions, detection of the disease at an early stage during regular health examinations can reduce both the incidence and mortality of the disease. Although sigmoidoscopy offers significant improvements in the detection rate of CRC, its diagnostic value is limited by its high costs and inconvenience. Therefore, there is a compelling need for the identification of noninvasive biomarkers that can enable earlier detection of CRC. Accordingly, many validation studies have been conducted to evaluate genetic, epigenetic or protein markers that can be detected in the stool or in serum. Currently, the fecal-occult blood test is the most widely used method of screening for CRC. However, advances in genomics and proteomics combined with developments in other relevant fields will lead to the discovery of novel non invasive biomarkers whose usefulness will be tested in larger validation studies. Here, noninvasive molecular biomarkers that are currently used in clinical settings and have the potential for use as CRC biomarkers are discussed.

Angiopoietin-1 prevents hypertension and target organ damage through its interaction with endothelial Tie2 receptor.

AIMS: The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage. METHODS AND RESULTS: To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules. CONCLUSION: The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.

Combined administration of naked DNA vectors encoding VEGF and bFGF enhances tissue perfusion and arteriogenesis in ischemic hindlimb.

Few studies have examined in detail the combined effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene delivery on collateral development. Here, we evaluated the potential synergism of naked DNA vectors encoding VEGF and bFGF using a skeletal-muscle based ex vivo angiogenesis assay and compared tissue perfusion and limb loss in a murine model of hindlimb ischemia. In the ex vivo angiogenesis assay, the VEGF+bFGF combination group had a larger capillary sprouting area than those of the LacZ, VEGF, and bFGF groups. Consistent with these results, regional blood flow recovery on day 14 was also highest in the VEGF+bFGF combination group, followed by the bFGF, VEGF, and LacZ groups. The limb loss frequency was 0% in the combination group, whereas the limb loss frequencies of the other groups were 7-29%. The ischemic muscles of the combination group revealed evidence of increased angiogenesis and arteriogenesis and the upregulated expression of genes that may be associated with arteriogenesis, such as those for cardiac ankyrin repeat protein, early growth response factor-1, and transforming growth factor-beta1. Our study has implications for the development of a combined gene therapy for the vascular occlusive diseases.

Interaction between Tie receptors modulates angiogenic activity of angiopoietin2 in endothelial progenitor cells.

OBJECTIVE: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. METHODS AND RESULTS: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. CONCLUSIONS: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.

Cardiac expression profiles of the naked DNA vectors encoding vascular endothelial growth factor and basic fibroblast growth factor.

We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for beta-galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were detected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 microg and 25 microg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for alpha-smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angiogenesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.

Pharmacokinetics and biodistribution of a pGT2-VEGF plasmid DNA after administration in rats.

Intramyocardial administration of gene therapy vectors expressing angiogenic factors have been attempted as an alternative to conventional surgical methods for the management of myocardial ischemia. In this study, we have developed the pGT2-VEGF, a plasmid DNA vector expressing human VEGF165, for the management of ischemic cardiovascular disease and investigated in vivo pharmacokinetics and tissue distribution of pGT2-VEGF after intramyocardial and intravenous administration in rats. A high concentration of pGT2-VEGF was observed in the heart after intramyocardial injection of 300 microg, which is in line with the assumption that direct intramyocardial delivery enables extended localization at the administration site. Leakage of the pGT2-VEGF to the blood circulation was observed after intramyocardial injection, with an area under the curve (AUC) of 3.8 microg min/mL, as compared with 37.3 microg min/mL after intravenous injection of the same dose. The pGT2-VEGF concentration in blood peaked at 5 minutes after intramyocardial administration and declined rapidly to undetectable levels by 2 hours post-administration. In tissue distribution studies, pGT2-VEGF peaked at 5 minutes post-administration in various organs but was undetectable at 2 hours in all organs except heart, lung, and liver. Taken together, the results suggest that intramyocardial-delivered pGT2-VEGF was degraded rapidly in vivo and mainly persisted in target tissues, the heart. In addition, intramyocardial-administered pGT2-VEGF was expressed for longer periods than the persistence of the pGT2-VEGF plasmid DNA in a target tissue. Therefore, a direct myocardial injection of pGT2-VEGF might be useful for local therapeutic angiogenesis.

Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization.

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPC-transplanted group exhibited significantly more monocytes/macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization.

Adenoviral-mediated delivery of early growth response factor-1 gene increases tissue perfusion in a murine model of hindlimb ischemia.

To test the hypothesis that overexpression of early growth response factor-1 (Egr-1) contributes to the revascularization of ischemic limbs, a constitutively active form of Egr-1 (Egr-1*) was made and evaluated in vitro and in vivo. Analyses of the transduced myocytes revealed significant upregulation of bFGF, PDGF-A, PDGF-B, IGF-II, and TGF-beta1. A coculture assay of the paracrine effects indicated that Ad-Egr-1* promoted proliferation and migration of endothelial cells. When Ad-Egr-1* was injected into the tibialis anterior muscle of mice, followed by explant culture in growth factor-reduced Matrigel, many capillary-like structures were observed in the Egr-1* group compared with minimal sprouting from the LacZ group, suggesting an angiogenic potential of Egr-1*. Next we evaluated Ad-Egr-1* in a murine model of hindlimb ischemia. Compared with slow revascularization in the control PBS or LacZ group, a rapid increase in tissue perfusion was observed in the Egr-1* group and the difference in flux ratio was statistically significant at day 7. In the injected muscle, expression of Egr-1*, upregulation of its target genes, and increased number of vessels staining positive for smooth muscle alpha-actin were observed. These results suggest that Egr-1 plays an important role in vascular recovery after occlusion and could be a potential target for therapeutic angiogenesis.

Long-term and sustained COMP-Ang1 induces long-lasting vascular enlargement and enhanced blood flow.

Vascular enlargement is a characteristic feature of angiopoietin-1 (Ang1)-induced changes in adult blood vessels. However, it is unknown whether tissues having Ang1-mediated vascular enlargement have more blood flow or whether the enlargement is reversible. We have recently created a soluble, stable and potent Ang1 variant, COMP-Ang1. In the present study, we investigated the effects of varied dose and duration of COMP-Ang1 on vascular enlargement and blood flow in the tracheal microvasculature of adult mice and explored a possible mechanism of long-lasting vascular enlargement. We found that COMP-Ang1 administered by adenoviral vector induced long-lasting vascular enlargement and increased tracheal blood flow. In contrast, short-term administration of COMP-Ang1 recombinant protein induced transient vascular enlargement that spontaneously reversed within a month. In both cases, the vascular enlargement resulted from endothelial proliferation. The COMP-Ang1-induced vascular remodeling is mediated mainly through Tie2 activation. Sustained overexpression of Tie2 could participate in the maintenance of vascular changes. Together, our findings indicate that sustained treatment with COMP-Ang1 can produce long-lasting vascular enlargement and increased blood flow.

Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung.

Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 microg/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.

Aldosterone upregulates connective tissue growth factor gene expression via p38 MAPK pathway and mineralocorticoid receptor in ventricular myocytes.

The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pattern in p38 MAPK. Blocking studies using pretreatment of the inhibitor of each pathway revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pretreatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.

C-reactive protein impairs angiogenic functions and decreases the secretion of arteriogenic chemo-cytokines in human endothelial progenitor cells.

C-reactive protein (CRP), a predictor of future cardiovascular diseases, has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation. This proatherogenic CRP was speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs), possibly impairing vascular regeneration and increasing cardiovascular vulnerability to ischemic injury. Herein, we investigated the direct effect of CRP on angiogenic activity and gene expression in human EPCs. Incubation of EPCs with human recombinant CRP significantly inhibited EPC migration in response to vascular endothelial growth factor, possibly by decreasing the expression of endothelial nitric oxide synthase and subsequent nitric oxide production. In addition, CRP-treated EPCs showed the reduced adhesiveness onto an endothelial cell monolayer. When assayed for the gene expression of arteriogenic chemo-cytokines, CRP substantially decreased their expression levels in EPC, in part due to the upregulation of suppressors of cytokine signaling proteins. These results suggest that CRP directly attenuates the angiogenic and possibly arteriogenic functions of EPCs. This CRP-induced EPC dysfunction may impair the vascular regenerative capacity of EPCs, thereby leading to increased risk of cardiovascular diseases.

Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease.

This phase 1 clinical trial tested the safety of intramuscular gene transfer by using naked plasmid DNA encoding the gene for VEGF, and analyzed the potential therapeutic benefits in patients with severe peripheral arterial disease (PAD). This study was an open-labeled, dose- escalating, single-center trial on nine male patients with severe debilitating PAD who had not responded to conventional therapy. Seven had Buerger's disease and two had arteriosclerosis obliterans. Plasmid DNA (pCK) containing human VEGF165 was given by eight intramuscular injections in and around the area in need of new blood vessels. The study evaluated three escalating total doses (2, 4, and 8 mug of pCK- VEGF165), with half of each total dose given four weeks apart. The follow-up duration was nine months. The gene injections were well tolerated without significant side effects or laboratory abnormalities related to gene transfer. Three patients showed transient edema in their extremities. Ischemic pain of the affected limb was relieved or improved markedly in six of seven patients. Ischemic ulcers healed or improved in four of six patients. The mean ankle-brachial index (ABI) improved significantly. Six of nine patients showed an increase in collateral vessels around the injection sites demonstrated by digital subtraction angiography. However, there was no relationship between the degree of ABI improvement and the dose given. Mean plasma levels of VEGF did not increase significantly. In conclusion, intramuscular injections of pCK- VEGF165 can be performed safely to induce therapeutic angiogenesis in patients with severe PAD.