Importance Of Vascular Endothelial Growth Factor Research Articles

MicroRNAs may provide new strategies in the treatment and diagnosis of diabetic retinopathy: Importance of VEGF

Diabetic retinopathy (DR) is ocular microvascular complications of diabetes mellitus. Along with the increasing prevalence of diabetes worldwide, DR has come into the major cause of human blindness. Several studies have demonstrated the important roles of the expression alteration in the proteins contributed to vascular dysfunction during DR, especially vascular endothelial growth factor (VEGF). However, there is a need for further mechanistic research in this context to design new therapeutic and diagnostic programs. MicroRNAs (miRNAs, miRs) have been introduced as key controllers of gene expression in a variety of biological processes including differentiation, proliferation, and metabolism. Altered expression of miRNAs during DR development indicates a close relationship between these regulatory molecules and DR through regulating gene expressions. This review discusses and updates the functions of miRNA-dependent pathways and key roles of VEGF in the DR, which may increase our understanding and ability to target these small but important molecules to efficiently improve therapeutic and diagnostic approaches.

Relationship between levels of vascular endothelial growth factor in blood and severity of diabetic retinopathy

Aim: The aim of the study was to establish the relationship between levels of vascular endothelial growth factor (VEGF) in blood and severity of diabetic retinopathy (DR). Background: The importance of VEGF in the pathogenesis of DR is evident from numerous studies demonstrating a significant increase in VEGF levels in samples obtained from the eye. However, a correlation between blood levels of VEGF and DR has not been conclusively proven or disproven. In this article, we demonstrate the relationship between blood levels of VEGF and the severity of DR. Materials and Methods: This is a hospital-based descriptive analytic study. The study population consisted of 75 Type 2 diabetic patients attending outpatient department for routine DR screening. After obtaining informed consent, 5 ml of blood was drawn from each patient and estimated for the levels of serum VEGF. The data thus obtained were correlated with the grade of DR. Additional parameters studied were duration of diabetes mellitus (DM), hemoglobin levels, blood urea, serum creatinine, fasting blood sugar (FBS) and random blood sugar (RBS), and hemoglobin A1c (HBA1c) levels. Statistical analysis used in this study was Chi-square test, Student's t-test, independent one sample t-test, and analysis of variance using SPSS software version. 19. Results: Overall mean VEGF levels for the study population of 75 diabetic patients were 577.01 ± 291.13 pg/ml. The average duration of DM in the study population was 7.2 years. The average hemoglobin level in the study population was 11.53 ± 2.00 g/dl. The mean urea levels in mg/dl of the whole population were 35.47 ± 25.80 mg/dl. The mean creatinine levels for the population were 1.42 ± 1.55 mg/dL. The mean RBS levels for the whole population were 228.95 ± 74.499 mg/dL. The mean FBS levels for the 75 patients were 165.87 ± 59.04 mg/dl. The mean HBA1c levels were 9.49 ± 2.37%. Conclusion: Levels of blood VEGF are elevated in diabetic patients regardless of whether they have DR or not. There is no statistically significant relationship between blood levels of VEGF and the severity of DR. Even though severe grades of DR showed more anemia, there is no statistically significant relationship between anemia and DR. The levels of urea and creatinine were elevated in the more severe grade of DR. VEGF, therefore, could present a potential treatment and preventive strategy for not only DR but also DM and its complications in general.

Abstract 26: Identifying The Role of and Treatment Targeting Bone Progenitor Cell VEGF Secretion On The Niche Supporting Traumatic Heterotopic Ossification

PURPOSE: Patients who sustain mechanical trauma, spinal cord injury, burns, or extremity surgeries are at risk for developing heterotopic ossification (HO), the pathologic formation of extraskeletal bone. HO is formed through a process of endochondral ossification initiated by acute inflammation. Vascular endothelial growth factor (VEGF) has been shown to be critical for both normal bone development and for fracture repair. We hypothesized that VEGF plays a central role during ossification of the cartilaginous matrix present during pathologic HO formation and that therapeutic targeting of the vascular niche is sufficient to prophylax against traumatic HO. METHODS: Male C57BL/6J mice underwent Achilles’ tendon transection and 30% of total body surface area (TBSA) dorsal burn injury to induce HO. Mice underwent Microfil CT and near infra-red imaging with intravascular injection of Angiosense to survey local vascularity. CDH5 endogenous reporter lines were imaged with confocal microscope. Hindlimb sections from injured mice were immunostained and injury site was harvested for flow cytometry and PCR. To further validate these findings and define the source of VEGF, co-staining with PDGFRa and VEGF was performed. Mice with genetic loss of Vegf in cells of mesenchymal lineage (Vegf cKO: Prx-cre/Vegffl/fl) and their littermate controls underwent burn/tenotomy (n=4–7). Human histological sections from analogous early HO sites were also examined via immunohistochemistry to confirm the translational value of these findings. Finally, a subset of mice after burn/tenotomy were treated with bevacizumab (biweekly injections of drug 10mg/kg) or vehicle control and were analyzed by histology (3 weeks) and MicroCT (9 weeks, n=5–6) for therapeutic testing. RESULTS: In wild-type mice, vascular density was intimately associated with the HO anlagen as demonstrated by near infrared imaging with Angiosense, Microfil imaging 5 weeks after burn/tenotomy, and endogenous CDH5 signal. Immunostaining of early human HO specimens with VEGFa and PDGFRa confirmed co-localization, corroborated by observations in mouse histology. Mice treated with bevacizumab, a potent anti-VEGF antibody, formed significantly less HO when compared at 9 weeks with vehicle control (2.64 v. 6.85 mm3, p=.0013). Genetic targeting produced parallel findings, with Vegf cKO mice forming significantly less HO near the distal tibia when compared with littermate controls (2.52 v. 4.53 mm3/mm, normalized to tibial cortical thickness compared to littermate control). CONCLUSIONS: Leveraging our knowledge of normal bone development, these findings suggest that VEGF from the mesenchymal niche plays a critical role in the formation of HO and can be successfully targeted to attenuate this process via bevacizumab, an FDA-approved pharmacologic agent used for its anti-VEGF properties. Given the importance of VEGF and angiogenesis during normal post-injury healing, future studies will focus on identifying the ideal treatment timing to prevent ossification of the cartilage anlagen and minimize off target effects. C. Hwang: None. S. Ucer: None. M. Sorkin: None. S. Loder: None. M.T. Chung: None. C. Pagani: None. J. Li: None. C. Priest: None. C. Breuler: None. K. Vasquez: None. S. Li: None. J. Xu: None. A. James: None. S. Agarwal: None. B. Levi: None.

Therapeutic effect of apatinib-loaded nanoparticles on diabetes-induced retinal vascular leakage.

Apatinib, a novel and selective inhibitor of vascular endothelial growth factor (VEGF) receptor 2, has been demonstrated recently to exhibit anticancer efficacy by inhibiting the VEGF signaling pathway. Given the importance of VEGF in retinal vascular leakage, the present study was designed to investigate whether apatinib-loaded polymeric nanoparticles inhibit VEGF-mediated retinal vascular hyperpermeability and block diabetes-induced retinal vascular leakage. For the delivery of water-insoluble apatinib, the drug was encapsulated in nanoparticles composed of human serum albumin (HSA)-conjugated polyethylene glycol (PEG). In vitro paracellular permeability and transendothelial electric resistance assays showed that apatinib-loaded HSA-PEG (Apa-HSA-PEG) nanoparticles significantly inhibited VEGF-induced endothelial hyperpermeability in human retinal microvascular endothelial cells. In addition, they substantially reduced the VEGF-induced junctional loss and internalization of vascular endothelial-cadherin, a major component of endothelial junction complexes. In vivo intravitreal injection of Apa-HSA-PEG nanoparticles in mice blocked VEGF-induced retinal vascular leakage. These in vitro and in vivo data indicated that Apa-HSA-PEG nanoparticles efficiently blocked VEGF-induced breakdown of the blood–retinal barrier. In vivo experiments with streptozotocin-induced diabetic mice showed that an intravitreal injection of Apa-HSA-PEG nanoparticles substantially inhibited diabetes-induced retinal vascular leakage. These results demonstrated, for the first time, that apatinib-loaded nanoparticles may be a promising therapeutic agent for the prevention and treatment of diabetes-induced retinal vascular disorders.

Effect of centrifugation time on growth factor and MMP release of an experimental platelet-rich fibrin-type product

Platelet-rich fibrin (PRF) has a controlled release of growth factors due to the fibrin matrix structure. Different centrifugation protocols were suggested for PRF preparation. Since the derivation method of PRF can alter its contents, in the present study it is aimed to investigate the cell contents and transforming growth factor beta-1 (TGF-β1), platelet-derived growth factor (PDGF-AB), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and-8 release from experimental PRF-type membranes obtained with different centrifugation times at 400 gravity. Three blood samples were collected from 20 healthy non-smoker volunteers. One tube was used for whole blood analyses. The other two tubes were centrifuged at 400 g for 10 minutes (group A) or 12 minutes (group B). Each experimental PRF-type membrane was placed in Dulbecco’s Modified Eagle’s Medium (DMEM)and at 1, 24 and 72 hours, TGF-β1, PDGF-AB, VEGF, MMP-1 and -8 release amounts were analysed by enzyme-linked immunosorbent assay (ELISA). The blood cell count of membranes was determined by subtracting plasma supernatant and red blood cell (RBC) mixture from the whole blood cell counts. At 72 hours, the VEGF level of group B was statistically higher than that of group A (p = 0.040). The centrifugation time was not found to influence the release of other growth factors, enzymes and cell counts. Within the limits of the present study, it might be suggested that centrifugation time at a constant gravity has a significant effect on the VEGF levels released from experimental PRF-type membrane. It can be concluded that due to the importance of VEGF in the tissue healing process, membranes obtained at 12-minute centrifugation time may show a superior potential in wound healing.

The Importance of Vascular Endothelial Growth Factor as a Marker of Angiogenesis and Lymphangiogenesis in Non-small Cell Lung Cancer

Abstract Lung cancer is the main cause of cancer death both in men and women. In spite of progress seen in the early diagnosis of lung cancer, and implementation of new treatment principles for these patients, 5 year survival of non-small cell lung cancer patients undergoing surgery is low. Introduction of anti-angiogenic therapy administered concomitantly with conventional chemotherapy agents represented practically the first success seen in the treatment of lung cancer in the last 20 years. The aim of this paper is to review the literature informations about the importance of VEGF (vascular endothelial growth factor) as a marker of angiogenesis in patients with non-small cell lung cancer. Therefore, we practiced a literature review about these topics: the importance of VEGF in tumor angiogenesis and lymphangiogenesis in patients with non-small cell lung cancer and his importance as a prognostic factor at these patients, the prognostic impact of serum levels of VEGF and of the cellular expression of VEGF at these patients and also we reviewed the value of the antiangiogenic therapy.

Delayed brain radiation necrosis: pathological review and new molecular targets for treatment.

Delayed radiation necrosis is a well-known adverse event following radiotherapy for brain diseases and has been studied since the 1930s. The primary pathogenesis is thought to be the direct damage to endothelial and glial cells, particularly oligodendrocytes, which causes vascular hyalinization and demyelination. This primary pathology leads to tissue inflammation and ischemia, inducing various tissue protective responses including angiogenesis. Macrophages and lymphocytes then infiltrate the surrounding areas of necrosis, releasing inflammatory cytokines such as interleukin (IL)-1α, IL-6, and tumor necrosis factor (TNF)-α. Microglia also express these inflammatory cytokines. Reactive astrocytes play an important role in angiogenesis, expressing vascular endothelial growth factor (VEGF). Some chemokine networks, like the CXCL12/CXCR4 axis, are upregulated by tissue inflammation. Hypoxia may mediate the cell-cell interactions among reactive astrocytes, macrophages, and microglial cells around the necrotic core. Recently, bevacizumab, an anti-VEGF antibody, has demonstrated promising results as an alternative treatment for radiation necrosis. The importance of VEGF in the pathophysiology of brain radiation necrosis is being recognized. The discovery of new molecular targets could facilitate novel treatments for radiation necrosis. This literature review will focus on recent work characterizing delayed radiation necrosis in the brain.

Aflibercept – Setting its Sights on Diabetic Macular Oedema

A satellite symposium entitled ‘Aflibercept* – Setting Its Sights On Diabetic Macular Oedema (DMO)’ was chaired by Jean-François Korobelnik and was convened at the 2014 European Association for Vision and Eye Research (EVER) Congress. The symposium discussed the science behind DMO, in particular, the role of vascular endothelial growth factor (VEGF) and associated inflammatory mechanisms that alter fluid transport from capillaries into retinal tissues leading to focal leakage, fluid accumulation, macular damage and eventual blindness. This discussion of the pathophysiology emphasised the importance of VEGF as a target for DMO treatments. Management of diabetes and prevention of progression of diabetic retinopathy leading to DMO requires strict control of glycated haemoglobin (HbA1c), blood pressure and lipid levels. Once DMO has developed and vision is impaired, the anti-VEGF agents have emerged as vital components of disease management and are becoming the first-line standard of care. Aflibercept (EYLEA®) and ranibizumab (Lucentis®) are approved agents for DMO and have shown significant efficacy in clinical trials in terms of visual acuity gains, decreased retinal thickness and have good safety profiles. The symposium finally focused on the use of aflibercept in DMO. In large-scale trials (VIVID and VISTA), this treatment has been compared head-to-head with laser treatment and during 1 year of treatment, showed substantial efficacy benefits, no new safety signals and the potential for lower frequency intravitreal dosing at 8- rather than 4-week intervals for monitoring andpro re natadosing.

High‐density lipoproteins augment hypoxia‐induced angiogenesisviaregulation of post‐translational modulation of hypoxia‐inducible factor 1α

Increasing evidence suggests that high-density lipoproteins (HDLs) promote hypoxia-induced angiogenesis. The hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway is important in hypoxia and is modulated post-translationally by prolyl hydroxylases (PHD1-PHD3) and E3 ubiquitin ligases (Siah1 and Siah2). We aimed to elucidate the mechanisms by which HDLs augment hypoxia-induced angiogenesis. Preincubation (16 h) of human coronary artery endothelial cells with reconstituted high-density lipoprotein (rHDL) containing apolipoprotein A-I (apoA-I) and phosphatidylcholine (20 μM, final apoA-I concentration), before hypoxia, increased Siah1 (58%) and Siah2 (88%) mRNA levels and suppressed PHD2 (32%) and PHD3 (45%) protein levels compared with hypoxia-induced control levels. After Siah1/2 small interfering RNA knockdown, rHDL was unable to suppress PHD2/3 and failed to induce HIF-1α, VEGF, and tubulogenesis in hypoxia. Inhibition of the upstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway also abrogated the effects of rHDL. Furthermore, knockdown of the scavenger receptor SR-BI attenuated rHDL-induced elevations in Siah1/2 and tubulogenesis in hypoxia, indicating that SR-BI plays a key role. Finally, the importance of VEGF in mediating the ability of rHDL to drive hypoxia-induced angiogenesis was confirmed using a VEGF-neutralizing antibody. In summary, rHDL augments the HIF-1α/VEGF pathway via SR-BI and modulation of the post-translational regulators of HIF-1α (PI3K/Siahs/PHDs). HDL-induced augmentation of angiogenesis in hypoxia may have implications for therapeutic modulation of ischemic injury.

The Role of Vascular Endothelial Growth Factor in Metastatic Prostate Cancer to the Skeleton

Despite the clinical implication and high incidence of bone and spinal metastases, the molecular mechanisms behind prostate cancer metastasis to bone and spine are not well understood. In this review the molecular mechanisms that may contribute to the highly metastatic phenotype of prostate cancer are discussed. Proangiogenic factors such as vascular endothelial growth factor (VEGF) have been shown to not only aid in the metastatic capabilities of prostate cancer but also encourage the colonization and growth of prostate tumour cells in the skeleton. The importance of VEGF in the complex process of prostate cancer dissemination to the skeleton is discussed, including its role in the development of the bone premetastatic niche, metastatic tumour cell recognition of bone, and bone remodeling. The expression of VEGF has also been shown to be upregulated in prostate cancer and is associated with clinical stage, Gleason score, tumour stage, progression, metastasis, and survival. Due to the multifaceted effect VEGF has on tumour angiogenesis, tumour cell proliferation, and bone destruction, therapies targeting the VEGF pathways have shown promising clinical application and are being investigated in clinical trials.

Pegaptanib Sodium in the Treatment of Neovascular Age-Related Macular Degeneration

Pegaptanib sodium is a polyethylene-glycolated, 28-nucleotide RNA aptamer that binds selectively to vascular endothelial growth factor (VEGF)165 but not smaller isoforms. Preclinical studies identified VEGF165 as an especially potent promoter of ocular neovascularization and inflammation. Following the pivotal clinical trials demonstrating the efficacy of intravitreal pegaptanib in treating all angiographic subtypes of neovascular age-related macular degeneration (NV-AMD), pegaptanib became the first anti-VEGF therapy to receive regulatory approval for this condition. In view of the importance of VEGF in a variety of tissues, including the cardiovascular system and the retina, concerns have been raised as to the risks that might accompany VEGF inhibition. It is thus of particular note that pegaptanib has proved to have a favorable safety record in treating NV-AMD, with no ocular or systemic safety signals having emerged over more than 4 years of clinical studies. Accordingly, in addition to its use as a single agent, pegaptanib has demonstrated promise in combinatorial regimens that employ nonselective anti-VEGF agents as an initial treatment followed by maintenance therapy with pegaptanib. Pegaptanib has also shown encouraging preliminary results in the treatment of diabetic macular edema, proliferative diabetic retinopathy, and macular edema secondary to retinal venous occlusive conditions.

The role of vascular endothelial growth factor SNPs as predictive and prognostic markers for major solid tumors

Angiogenesis is crucial for development and metastasis of tumors, and vascular endothelial growth factor (VEGF) is a key mediator of this process. The importance of VEGF in tumorigenesis and tumor progression makes it an attractive target for the development of anticancer therapies. Inhibition of angiogenesis has shown promising clinical efficacy; however, not all patients treated with antiangiogenic agents derive benefit from them. Some patients are predisposed to refractory disease, whereas others develop resistance after initial response. Patients may also have different severity of drug-related adverse events. Optimization of drug administration based on disease status and individual responsiveness is important in limiting the treatment failure and minimization of side-effects. Single nucleotide polymorphisms (SNP) in VEGF may alter VEGF protein concentrations, influence the process of angiogenesis, and may relate to interindividual variation in the risk and progression of selected tumors, and their resistance to treatments. This review examines the role of SNPs in the VEGF gene as predictive and prognostic markers for major solid tumors, including the breast, non-small cell lung, colorectal, and prostate cancers. Selected VEGF SNPs seem to be associated with risk of these cancers; however, there is lack of unanimity in findings, in part influenced by differences in study design and analysis.

VEGF serum concentrations in patients with long bone fractures: A comparison between impaired and normal fracture healing

Vascular endothelial growth factor (VEGF) plays an important role in the bone repair process as a potent mediator of angiogenesis and it influences directly osteoblast differentiation. Inhibiting VEGF suppresses angiogenesis and callus mineralization in animals. However, no data exist so far on systemic expression of VEGF with regard to delayed or failed fracture healing in humans. One hundred fourteen patients with long bone fractures were included in the study. Serum samples were collected over a period of 6 months following a standardized time schedule. VEGF serum concentrations were measured. Patients were assigned to one of two groups according to their course of fracture healing. The first group contained 103 patients with physiological fracture healing. Eleven patients with delayed or nonunions formed the second group of the study. In addition, 33 healthy volunteers served as controls. An increase of VEGF serum concentration within the first 2 weeks after fracture in both groups with a following decrease within 6 months after trauma was observed. Serum VEGF concentrations in patients with impaired fracture healing were higher compared to the patients with physiological healing during the entire observation period. However, statistically significant differences were not observed at any time point between both groups. VEGF concentrations in both groups were significantly higher than those in controls. The present results show significantly elevated serum concentrations of VEGF in patients after fracture of long bones especially at the initial healing phase, indicating the importance of VEGF in the process of fracture healing in humans.

Quantitative Analysis of Gene Expression for Vascular Endothelial Growth Factor and Its Application.

Vascular endothelial growth factor (VEGF) is a crucial mediator of angiogenesis, and plays an important role in pathogenesis of leukemia. However, the importance of VEGF in differentiation or apoptosis of leukemic cells remains to be evaluated. A competitor DNA fragment template of VEGF gene mimic was constructed with the method of gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) for analyzing VEGF gene expression was performed to assess the regulation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of an acute promyelocytic leukemia cell line NB4. In construction of a standard curve from which the amount of target cDNA was derived, serial dilutions of the target were co-amplified with a constant amount of mimic, and the intensities of bands corresponding to the target and the mimic were measured. CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. cQRT-PCR was a sensitive and reliable tool for analysis of VEGF gene expression, with a detectable range from 1 to 2 times 10 the fifth power molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3, 12.6, 3.6, and less than 1.0 times 10 the fifth powder per microgram of NB4 total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. The rapid down-regulation of VEGF gene expression during ATRA-induced NB4 cell differentiation was accompanied by an upregulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR could be used as an efficient method of qualitative analysis of VEGF gene expression. ATRA significantly depresses VEGF expression and its antileukemic effect can be brought through the two ways of differentiation induction and angiogenesis inhibition.

Combination of pegylated-liposomal doxorubicin (PLD) and bevacizumab (B) (PLD-B) in sarcoma (SAR)

20506 Background: Vascular endothelial growth factor (VEGF) plays an important role in many tumors including SAR. Because of the known efficacy of PLD in SAR and the presumed importance of VEGF in SAR, we have treated SAR patients with PLD-B. It is possible that the addition of B to PLD might alter the toxicity profile. The toxicities of this combination are not well described. Methods: To better define the toxicity of the combination of PLD-B in SAR, we reviewed our experience with PLD-B in patients treated between 2004 and 2006. Results: We identified 20 patients with SAR treated with PLD-B at our institution. There were 9 men and 11 women, with a median age of 47.5 years (range 23–77). Ten patients initiated therapy with PLD q28 days at a median dose of 45 mg/m2 (range 45–50), and 10 q14 days at a median dose of 21.2 mg/m2 (range 20–25), in combination with B at 5 mg/kg q14 days. A median of 5 cycles was given (range 1–15). Dose delay was required in 3 patients for the second cycle, and dose reduction was required in 9 patients by cycle 3. Of the 10 patients starting with monthly PLD, 7 were changed to q14 day PLD by cycle 4. Mucositis and skin toxicity were the dose limiting toxicities (DLT) in 14/20 patients, and myelosuppression was the DLT in 1/20 patients. Nine patients were felt to have clinical benefit; 3 had a PR and 6 had SD. Notably, 2/4 recurrent Ewing sarcoma patients had symptomatic improvement and at least SD for 10 and 11 months, respectively, with minor toxicity allowing a good quality of life. Conclusions: This report describes the toxicity profile of PLD-B in 20 patients with SAR. The toxicities observed were similar to those seen with PLD alone, however, these toxicities appeared more pronounced with the addition of B. Since VEGF is important for wound healing, the known effects of B on this process may contribute to more skin and mucosal toxicity of PLD at a given dose. Future studies of PLD-B should consider the potential of increased mucosal and skin toxicity. The use of q14 day PLD to allow more control over toxicity is suggested; a maximal starting dose of 20 mg/m2 is reasonable, although dose reduction will be common due to skin or mucosal toxicity. This combination can be given with limited toxicity, and meaningful responses were observed in recurrent sarcomas, including 2/4 Ewing sarcomas. No significant financial relationships to disclose.

Combination of bevacizumab (A) and pegylated-liposomal doxorubicin (PLD) (PLD-A) in sarcoma (SAR)

9556 Background: Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis (AG) in many tumors including SAR. A has demonstrated efficacy in several common solid tumors. Because of the known efficacy of PLD in SAR and the presumed importance of VEGF in SAR, we have treated SAR patients with PLD-A. It is possible that the addition of A to PLD might alter the toxicity profile. To our knowledge, the toxicities of this combination have not been previously described. Methods: To better define the toxicity of the combination of PLD-A in SAR, we reviewed our experience with PLD-A in patients with SAR treated between 2004 and 2005. Results: We identified 12 patients with SAR treated with PLD-A at our institution. There were 5 men and 7 women, with a median age of 43.5 years (range 27–57). Nine patients initiated therapy with PLD on a 28 day interval at a median dose of 45 mg/m2 (range 45–50), and 3 on a 14 day interval at a dose of 22.5 mg/m2, in combination with A at 5 mg/kg every 2 weeks. A median of 3 cycles was given (range 2–9). Standard CALGB response and NIH common toxicity criteria were used. Dose delay was required in 3 patients for the second cycle, and dose reduction was required in 5 patients by cycle 3. Of the 10 patients starting with monthly PLD, 6 were changed to q 2 week PLD by cycle 4. Mucositis and skin toxicity were the dose limiting toxicities (DLT) in 9/12 patients, and myelosuppression was the DLT in 1/12 patients. Conclusions: This report describes the toxicity profile of PLD-A in 12 patients with SAR. The toxicities observed were similar to those seen with PLD alone, however, there was a suggestion that these toxicities may be more pronounced with the addition of A, though this appears to vary among patients. Since VEGF is important for wound healing, the known effects of A on this process may contribute to more skin and mucosal toxicity of PLD at a given dose. Future studies of PLD-A should consider the potential of increased mucosal and skin toxicity, and the use of q2week PLD to allow more control over toxicity is suggested. [Table: see text]

Inhibition of vascular endothelial growth factor receptor 2 activity in experimental brain contusions aggravates injury outcome and leads to early increased neuronal and glial degeneration

Angiogenesis following traumatic brain injuries (TBIs) may be of importance for post-traumatic reparative processes and the development of secondary injuries. We have previously shown expression of vascular endothelial growth factor (VEGF), a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and VEGF receptors (VEGFR1 and 2) after TBI in rat. In the present work we tried to further elucidate the role of VEGF after TBI by performing specific VEGFR2 activity inhibition. In rats subjected to VEGFR2 blockage we report an increased haemorrhagic area (P < 0.05), early increase in serum levels of neural injury marker neuron-specific enolase (P < 0.05) and glial injury marker S100beta (P < 0.05), and increased numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling- (TUNEL-) and FluoroJade B- (P < 0.05) positive cells, all increases preceding the known VEGF/VEGFR vascular response in brain trauma. An increase in lesion area, as measured by decreased microtubuli-associated protein 2 expression (P < 0.05) and increased glial fibrillary acidic protein reactivity (P < 0.05), could also be demonstrated. In addition, vascular density, as measured by von Willebrandt factor-positive cells, was decreased (P < 0.05). No differences in post-traumatic inflammatory response, as measured by stainings for macrophages, granulocytes and intracellular adhesion molecules, were shown between the groups. Taken together, our findings point towards VEGF/VEGFR2 up-regulation after TBI as being an important endogenous cytoprotective mechanism in TBI. The possible importance of VEGF on the vascular, neuronal and glial compartments of the neurovascular unit after TBI is discussed.

Direct regulation of prostate blood flow by vascular endothelial growth factor and its participation in the androgenic regulation of prostate blood flow in vivo.

Previous studies on prostate blood flow regulation have indicated that androgen regulates prostate blood flow. However, the mechanism responsible for this regulation is unknown. In the present study, we focused on the effects of vascular endothelial growth factor (VEGF), a key factor responsible for angiogenesis and androgenic blood flow regulation. We examined in vivo the effect of VEGF on prostate blood flow and its participation in the androgenic regulation of this blood flow using a castrated rat model following subcapsular intraprostatic injection method. We found that VEGF is involved in blood flow regulation with an activity equal to that of dihydrotestosterone (DHT). The effect of VEGF on prostate blood flow was already seen at 30 min after the administration. The elevating effect of DHT on castrated rat prostate blood flow was abolished by coadministration of DHT with neutralizing anti-VEGF antibody. The change in VEGF-A mRNA expression in response to androgen stimulation was examined by double-fluorescent probe quantitative PCR (Taqman PCR). The results showed that androgenic regulation of VEGF gene expression occurred shortly after androgen stimulation. VEGF gene up-regulation was abolished or down-regulated by coadministration of neutralizing anti-VEGF antibody. This is the first report on the importance of VEGF in the androgenic regulation signaling pathway that affects prostate blood flow. Alternative treatment targeted toward anti-VEGF activity as a substitute for ordinary antiandrogenic therapy may be effective against prostate diseases, especially those with androgen-independent and hyperhemorrhagic status.

VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis.

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B, a new VEGF family member that binds to the tyrosine kinase receptor flt-1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF-B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF-B and its receptor flt-1 by ribonuclease protection assay and the pattern of VEGF-B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt-1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF-B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF-B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF-B and flt-1 (p=0.2) or tumour vascularity (p=0.4). VEGF-B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF-B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF-B may contribute to tumour progression by a non-angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF-B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti-VEGF receptor therapy targeted to flt-1 (VEGFR1) as well as kdr (VEGFR2).

Immunohistochemical measurement of tumor vascular endothelial growth factor in breast cancer. A more reliable predictor of tumor stage than microvessel density or serum vascular endothelial growth factor.

Microvessel density counting is commonly proposed as a method of assessing angiogenesis. However, results have been difficult to reproduce because of many methodological inconsistencies. Vascular endothelial growth factor (VEGF), an angiogenic regulator, is also a poor prognostic indicator in breast cancer, correlating in many studies with microvessel density. In this study, VEGF and microvessel density counting were examined as methods of assessing angiogenesis in breast cancer and correlated with tumor stage. A representative tumor section was stained with anti-CD34 and anti-VEGF. Microvessel density was evaluated using the manual "hot-spot" procedure and a semiquantitative image analysis system. Serum VEGF levels were available from an additional nine patients. Results were analyzed using Kendall's tau correlation. Tumor stage correlated with tumor VEGF, but not with microvessel "hot-spot" or vessel counts. There was no correlation between scores obtained from the manual or semiquantitative methods. Serum VEGF did not correlate with either tumor VEGF or tumor stage. The prognostic importance of VEGF in invasive breast cancer is associated with tumor stage. Measurement of tumor VEGF, as an indicator of angiogenesis, is more reliable prognostically than measurement of microvessel density or serum VEGF. Routine measurement of microvessel density in breast cancer is less reliable.