Seyedhossein Aharinejad

Colony-Stimulating Factor-1 Blockade by Antisense Oligonucleotides and Small Interfering RNAs Suppresses Growth of Human Mammary Tumor Xenografts in Mice

Colony-stimulating factor (CSF)-1 is the primary regulator of tissue macrophage production. CSF-1 expression is correlated with poor prognosis in breast cancer and is believed to enhance mammary tumor progression and metastasis through the recruitment and regulation of tumor-associated macrophages. Macrophages produce matrix metalloproteases (MMPs) and vascular endothelial growth factor, which are crucial for tumor invasion and angiogenesis. Given the important role of CSF-1, we hypothesized that blockade of CSF-1 or the CSF-1 receptor (the product of the c-fms proto-oncogene) would suppress macrophage infiltration and mammary tumor growth. Human MCF-7 mammary carcinoma cell xenografts in mice were treated with either mouse CSF-1 antisense oligonucleotide for 2 weeks or five intratumoral injections of either CSF-1 small interfering RNAs or c-fms small interfering RNAs. These treatments suppressed mammary tumor growth by 50%, 45%, and 40%, respectively, and selectively down-regulated target protein expression in tumor lysates. Host macrophage infiltration; host MMP-12, MMP-2, and vascular endothelial growth factor A expression; and endothelial cell proliferation within tumors of treated mice were decreased compared with tumors in control mice. In addition, mouse survival significantly increased after CSF-1 blockade. These studies demonstrate that CSF-1 and CSF-1 receptor are potential therapeutic targets for the treatment of mammary cancer.

Colony-Stimulating Factor-1 Antibody Reverses Chemoresistance in Human MCF-7 Breast Cancer Xenografts

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol-linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringers solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer-related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer.

Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.

Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A165, VEGF-A189, and VEGF-B and the protein level of VEGF-A and VEGF-R1 were downregulated compared with controls (P <0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls (P <0.05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen (P <0.05). Blunted VEGF-A and VEGF-R1 protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A165, to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Colon Cancer Cell–Derived Tumor Necrosis Factor-α Mediates the Tumor Growth–Promoting Response in Macrophages by Up-regulating the Colony-Stimulating Factor-1 Pathway

The interplay between malignant and stromal cells is essential in tumorigenesis. We have previously shown that colony-stimulating factor (CSF)-1, matrix metalloprotease (MMP)-2, and vascular endothelial growth factor (VEGF)-A production by stromal cells is enhanced by CSF-1-negative SW620 colon cancer cells. In the present study, the mechanisms by which colon cancer cells up-regulate host factors to promote tumorigenesis were investigated. Profiling of tumor cell cytokine expression in SW620 tumor xenografts in nude mice showed increased human tumor necrosis factor (TNF)-alpha mRNA expression with tumor growth. Incubation of macrophages with small interfering (si) RNAs directed against TNF-alpha or TNF-alpha-depleted SW620 cell conditioned medium versus SW620 cell conditioned medium failed to support mouse macrophage proliferation, migration, and expression of CSF-1, VEGF-A, and MMP-2 mRNAs. Consistent with these results, human TNF-alpha gene silencing decreased mouse macrophage TNF-alpha, CSF-1, MMP-2, and VEGF-A mRNA expression in macrophages cocultured with human cancer cells. In addition, inhibition of human TNF-alpha or mouse CSF-1 expression by siRNA reduced tumor growth in SW620 tumor xenografts in mice. These results suggest that colon cancer cell-derived TNF-alpha stimulates TNF-alpha and CSF-1 production by macrophages, and that CSF-1, in turn, induces macrophage VEGF-A and MMP-2 in an autocrine manner. Thus, interrupting tumor cell-macrophage communication by targeting TNF-alpha may provide an alternative therapeutic approach for the treatment of colon cancer.

Novel Insights Into the Mechanisms and Treatment of Intramural Hematoma Affecting the Entire Thoracic Aorta

BACKGROUND The purpose of this study was to address a previously not described mechanism underlying intramural hematoma (IMH) of the entire thoracic aorta and to test the hypothesis whether endovascular stent graft placement in this particular mechanism could be beneficial. METHODS Within a 5-year period, we treated 8 patients with IMH affecting the entire thoracic aorta. The presumed site of initial plaque rupture was chosen as target for endovascular stent graft placement. RESULTS In all patients, a small atherosclerotic plaque at the free lateral wall or at the concavity of the distal aortic arch could be identified as initial site of IMH. Endovascular stent graft placement was performed successfully in all patients. By covering the suspected primary lesion, resorption of IMH especially within the ascending aorta could be achieved. Mean follow-up is 16 months (range, 1 to 25). CONCLUSIONS Plaque rupture may be identified as the cause of IMH in a previously unrecognized subgroup of patients. If at the convexity of the distal arch, supra-aortic branches prevent retrograde extension toward the ascending aorta. If at the free lateral wall or at the concavity, IMH may affect the entire thoracic aorta, owing to the lack of the natural barrier of the supra-aortic branches. Endovascular stent graft placement of this plaque-associated IMH may be more effective and less invasive than conventional surgery to treat the entire thoracic aortic disease.

Renal Vein Anatomy and its Implications for Retroperitoneal Surgery

Because of its complicated embryological development, the anatomy of the renal veins shows extensive variability. A full understanding of the potential anatomical variations is imperative for retroperitoneal operations. Based on 4,520 retroperitoneal computerized tomography scans, anatomical studies of autopsy material of 354 unselected cases and intraoperative observations made during 215 major retroperitoneal procedures, an attempt was made to define the most common renal vein variants and retrace their development during embryogenesis. Awareness of rare anomalies in urological and general surgery is crucial to prevent severe damage to the venous drainage of the left kidney, and because troublesome bleeding may occur during vascular and retroperitoneal oncological procedures in patients with unknown venous anomalies. We found the incidence of these variants to be 0.8 versus 1.7 versus 3.7%, respectively.

Elevated transforming growth factor-beta 1 (TGF-β1) levels in human fracture healing

Introduction Transforming growth factor-beta 1(TGF-β1) is a regulatory protein, involved in bone fracture healing. Circulating TGF-β1 levels have been reported to be a predictor of delayed bone healing and non-union, suggesting active relationship between tissue and circulating TGF-β1 in fracture healing. The purpose of this study was to analyse TGF-β1 local and serum concentrations in fracture healing to further contribute to the understanding of molecular regulation of fracture healing. Patients and methods Serum samples of 113 patients with long bone fractures were collected over a period of 6 months following a standardised time schedule. TGF-β1 serum concentrations were measured using ELISA. Patients were assigned to 2 groups: Group 1 contained 103 patients with physiological healing. Group 2 contained 10 patients with impaired healing. Patients in both groups were matched. One patient of the group 2 had to be excluded because of missing match partner. In addition, fracture haematoma from 11 patients of group 1 was obtained to analyse local TGF-β1 concentrations. 33 volunteers donated serum which served as control. Results TGF-β1 serum concentrations increased during the early healing period and were significantly higher in patients with physiological healing compared to controls (P = 0.04). Thereafter, it decreased continuously between weeks 2 and 8 and fell again after week 8. TGF-β1 serum concentrations in patients with physiological healing were significantly higher at week 24 compared to controls (P = 0.05). In non-unions, serum concentrations differed significantly from those of controls at week 6 (P = 0.01). No significant difference in between patients with physiological and impaired fracture healing was observed. Fracture haematoma contained significantly higher TGF-β1 concentrations than peripheral serum of the patients (P = 0.017). Conclusion Elevated levels of TGF-β1 in haematoma and in serum after bone fracture especially during the entire healing process indicate its importance for fracture healing.

Direct epicardial shock wave therapy improves ventricular function and induces angiogenesis in ischemic heart failure

OBJECTIVES Direct application of low-energy unfocused shock waves induces angiogenesis in ischemic soft tissue. The potential effects of epicardial shock wave therapy applied in direct contact to ischemic myocardium are uncertain. METHODS For induction of ischemic heart failure in a rodent model, a left anterior descending artery ligation was performed in adult Sprague-Dawley rats. After 4 weeks, reoperation with (treatment group, n = 60) or without (control group, n = 60) epicardial shock wave therapy was performed. Low-energy shock waves were applied in direct contact with the infarcted myocardium (300 impulses at 0.38 mJ/m(2)). Additionally, healthy animals (n = 30) with normal myocardium were studied. Angiogenesis, ventricular function upregulation of growth factors, and brain natriuretic peptide levels were analyzed. RESULTS Histologic analysis revealed significant angiogenesis 6 weeks (treatment group: 8.2 +/- 3.7 vs control group: 2.9 +/- 1.9 vessels per field, P = .016) and 14 weeks (treatment group: 7.1 +/- 3.1 vs control group: 3.2 +/- 1.8 vessels per field, P = .011) after shock wave treatment. In the treatment group ventricular function improved throughout the follow-up period (6 weeks: 37.4% +/- 9% [P < .001] and 14 weeks: 39.5% +/- 9% [P < .001]). No improvement of ventricular function was observed in the control group (6 weeks: 28.6% +/- 5% and 14 weeks: 21.4% +/- 5%). Rat brain natriuretic peptide 45 levels were lower in the treatment group compared with those in the control group 6 and 14 weeks after treatment. Vascular endothelial growth factor, Fms-related tyrosine kinase 1, and placental growth factor levels were upregulated after 24 and 48 hours and 7 days in the treatment group. No effects on healthy myocardium were observed. CONCLUSION Direct epicardial low-energy shock wave therapy induces angiogenesis and improves ventricular function in a rodent model of ischemic heart failure.

VEGF-A and -C but not -B mediate increased vascular permeability in preserved lung grafts

Background. Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor, expressed in the lung. Overexpression of VEGF is associated with increased vascular permeability in the early stage of acute lung injury in mice. The role of various forms of VEGF in transplantation-induced lung injury is not well understood. Methods. VEGF mRNA and protein expression was measured in biopsies of preserved donor lung grafts as well as in control lung biopsies, using real-time reverse transcriptase–polymerase chain reaction and Western blot analysis. VEGF tissue expression was also evaluated by immunocytochemistry. Serum VEGF was measured in recipients after transplantation and in controls using ELISA. Results. Although VEGF-A and VEGF-C protein expression was up-regulated, their mRNA levels were decreased in donor versus control lung biopsies (P <0.05). VEGF-B mRNA was decreased, but its protein level was unchanged in donors. Flt-1 was unchanged, KDR gene expression was down-regulated in donors (P <0.05), and both receptors’ protein expression was under the detection level in donor and control lungs. VEGF-A was detected in pulmonary vessels and bronchi, whereas VEGF-C was only detectable in vessels of both donor and control lungs. After transplantation, serum VEGF increased (P <0.05) and returned to control baseline levels 12 weeks after surgery. Wet-to-dry lung weight was increased in donor versus control lungs. Conclusions. These results indicate that unventilated hypoxia increases vascular permeability in lung grafts and that this process is mainly regulated at VEGF-A and VEGF-C translational but not transcriptional level. Selective VEGF antagonism during graft preservation might be of benefit to counteract edema formation.

Fibrin-embedded administration of VEGF plasmid enhances skin flap survival.

The aim of the present study was to experimentally evaluate whether topical fibrin‐mediated administration of a vascular endothelial growth factor (VEGF)‐A plasmid to the wound bed can protect skin flaps from necrosis. A plasmid expression vector containing the VEGF‐A cDNA was constructed. The plasmid was then administered to the wound bed of rat abdominal skin flaps in a fibrin sealant. The percentage of viable, ischemic and necrotic tissue was assessed postoperatively as a baseline and after 3 and 7 days using digital surface area morphometry. Laser Doppler imaging of the flaps and VEGF‐A Western blot analysis of flap tissue were performed to assess angiogenesis and VEGF‐A tissue levels. Flaps treated with VEGF plasmids in the presence of uptake enhancing Lipofectamine transfection reagent increased flap survival 7 days postoperatively significantly associated with markedly elevated tissue perfusion and enhanced tissue VEGF‐A protein expression. Our results indicate that topical fibrin‐mediated administration of a VEGF‐A plasmid may serve as an alternative to previous strategies in treating ischemic skin flaps. The suggested therapeutic approach is easily applicable and inexpensive in preparation. Thus, this protocol may also enhance wound healing in posttrauma skin lacerations or in skin grafts.