Canine Vascular Endothelial Growth Factor Research Articles

In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma.

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

In-vitro binding analysis of anti-human vascular endothelial growth factor antibodies bevacizumab and aflibercept with canine, feline, and equine vascular endothelial growth factor

PurposePromising results have been described for antibodies binding vascular endothelial growth factor (VEGF) in patients with corneal neovascularization. Whether veterinary patients would also benefit from this therapeutic approach has not been investigated yet. We examined binding properties of anti-human VEGF antibodies bevacizumab (Avastin®) and aflibercept (Zaltrap®) for canine, feline, and equine VEGF. MethodsHuman, equine, feline, and canine VEGF were analyzed for sequence similarity using the “Basic Local Alignment Search Tool” (BLAST). Western-blot analysis and ELISA were used to assess binding properties. ResultsBLAST analysis revealed a sequence homology of canine, feline, and equine VEGF to human VEGF-A of 93%, 92%, and 89%, respectively. Western-blot analysis showed immunoreactivity of bevacizumab with human, canine, and feline VEGF, but not with equine VEGF. Aflibercept recognized VEGF of all tested species. ELISA data indicated that bevacizumab and aflibercept bind canine VEGF in a dose-dependent manner. Feline VEGF was bound by bevacizumab and aflibercept in a dose-independent manner. ELISA study further confirmed the lack of bevacizumab binding to equine VEGF, and yielded also a dose-independent binding by aflibercept. ConclusionsBevacizumab and aflibercept turned out to bind VEGF with species-specific differences. Further studies are required to investigate their efficacy and safety under clinical conditions.

Elevated Vascular Endothelial Growth Factor (VEGF) levels in the blood serum of dogs with malignant neoplasms of the oral cavity

Angiogenesis plays an essential role in the development of a neoplastic tumour by conditioning both its growth and the formation of metastases. The induction of blood vessel growth occurs under the influence of proangiogenic factors, among which Vascular Endothelial Growth Factor (VEGF) seems to be the most important. The aim of this research was to study the level of VEGF measured by ELISA in the serum of dogs with neoplasms of the oral cavity. The study material comprised samples of neoplastic tissue from 17 operated dogs and the serum of the examined animals as well as of dogs from the control group. The tissue samples were taken from dogs of different breeds, aged 6-14 years. The tumour type was determined in accordance with the applicable WHO classification. Blood samples taken from sick dogs and from animals of the control group were centrifuged, and immunoenzymatic labelling of VEGF was performed in the obtained serum using ELISA and R&D system reagents (Quantikine Canine VEGF). All stages of VEGF labelling were performed according to the recommendation of the test manufacturer. The median of VEGF in the serum of the dogs with neoplasms of the oral cavity was 40.64 pg/mL. The lowest value of 14.26 pg/mL was observed in the case of fibrosarcoma, and the highest value of 99.19 pg/mL in the case of squamous cell carcinoma. The VEGF median in the control group amounted to 11.14 pg/mL whereas the VEGF value in the groups of animals diagnosed with benign tumours ranged between 2.30 and 19.74 pg/mL. Elevated VEGF in the blood serum, in comparison with the benign tumour group and the control group, was observed in all examined neoplasms of the oral cavity. It was suggested that overexpression of VEGF can have a prognostic value and is useful in the early detection of neoplasms.

Serum vascular endothelial growth factor concentration in dogs diagnosed with chronic superficial keratitis

The objective of this study was to measure the vascular endothelial growth factor (VEGF) levels in dogs diagnosed with chronic superficial keratitis (CSK). The study was performed on 25 German shepherds (14 males and 11 females, aged between 3 and 11 years). The VEGF levels were determined in blood serum using commercially available enzyme-linked immunosorbent assay (ELISA; Quantikine Canine VEGF Immunoassay, R&D Systems). The test group of affected German shepherds was subdivided into two subgroups, based on the area of corneal neovascularisation. The first subgroup (9 patients) comprised dogs with neovascularisation observed in 1 to 2 quadrants of the right and left cornea, while the second subgroup (16 patients) comprised dogs with neovascularisation observed in 3 to 4 quadrants of the right and left cornea. The control group comprised 12 clinically healthy German shepherds (7 males and 5 females, aged between 3 and 9 years). The results were then statistically analysed by the Mann-Whitney test. The study indicated that the median serum VEGF concentration in healthy dogs was 14.9 pg/mL. The VEGF level observed in sick German shepherds was elevated (19.5 pg/mL) as compared to the values found in healthy dogs; however, a statistically significant increase in VEGF concentration, as compared to the values observed in healthy dogs, was only noted in the first subgroup, where the median VEGF concentration was 22.0 pg/mL. Elevated serum VEGF concentration was observed in German shepherds diagnosed with CSK. A statistically significant increase in VEGF levels was observed in dogs in the first stage of the disease, i.e. the early stage of neovascularisation.

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.

Aqueous humor vascular endothelial growth factor in dogs: association with intraocular disease and the development of pre‐iridal fibrovascular membrane

To examine the concentrations of vascular endothelial growth factor (VEGF) in aqueous humor of dogs with intraocular disease and to evaluate the association of aqueous humor VEGF with pre-iridal fibrovascular membrane (PIFM) formation. Two hundred and twenty six aqueous humor samples and 101 plasma samples were collected from 178 dogs with a variety of intraocular diseases (including cataract, primary glaucoma, uveitic glaucoma, aphakic/pseudophakic glaucoma, retinal detachment, lens luxation, and intraocular neoplasia), and aqueous humor was collected from 13 ophthalmically normal control dogs. Systemic disease status and administration of select medications were recorded. Aqueous and plasma VEGF was assayed via enzyme-linked immunosorbant assay validated for canine VEGF. Available histopathology samples were examined for the presence of PIFM. Where present, PIFMs were categorized as none, cellular, vascular or fibrous, and fibrovascular. Data were evaluated by mixed model anova, with application of Tukey-Kramer adjustment for multiple comparisons. There was no association between aqueous humor and plasma VEGF levels. Compared with normal controls, aqueous humor VEGF was significantly elevated for all intraocular diseases (P < 0.05) except for primary and diabetic cataracts. Systemic disease and administered medications had no significant effect on aqueous humor VEGF concentration. Compared to dogs without PIFM, aqueous humor VEGF was significantly higher in dogs with fibrovascular PIFM (P = 0.001), but not cellular (P = 0.1704) or fibrous/vascular PIFM (P = 0.0667). These findings confirm that VEGF is commonly elevated in aqueous humor of dogs with intraocular disease and likely plays a role in the causation or progression of a variety of intraocular disorders, including the development of PIFM.

Vascular endothelial growth factor in diabetic and nondiabetic canine cataract patients

To measure vascular endothelial growth factor (VEGF) levels in aqueous humor, serum, and plasma in diabetic and nondiabetic cataractous dogs. Canine VEGF was assayed in the plasma and serum of 32 dogs (20 diabetics; 12 nondiabetics) and aqueous humor in 57 eyes of those dogs (39 diabetic; 18 nondiabetic) undergoing phacoemulsification, using a commercial canine VEGF assay. Statistical analysis was performed using Fisher's PLSD, t-test, and regression analysis to compare values by diabetic status, duration of diabetes, age, weight, gender, left vs. right eye, and blood clarity. Plasma, but not serum or aqueous humor VEGF values of diabetics were significantly greater than nondiabetics (P = 0.019). Older nondiabetics (10-15 years) had higher plasma VEGF values than younger (0-5 and 5-10 years) dogs (P = 0.0002 and 0.0001, respectively). There was no significant difference in aqueous humor VEGF between left and right eyes in all patients. Serum and plasma, but not aqueous humor, VEGF values in females were significantly higher than males in both groups. Similar to human diabetic patients, VEGF aqueous humor values in all dogs are significantly higher than blood values. Aqueous humor VEGF values in human diabetics are elevated and correlate with the severity of diabetic retinopathy. However, aqueous humor values of VEGF in diabetic dogs are not greater than nondiabetics and may serve to protect the dog against development of diabetic retinopathy.

Effect of radiation on vascular endothelial growth factor expression in the C2 canine mastocytoma cell line

To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2. Canine mastocytoma cell line C2. C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter. C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation. The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.

Vascular Endothelial Growth Factor mRNA Expression and Peritumoral Edema in Canine Primary Central Nervous System Tumors

Vascular endothelial growth factor (VEGF) is an important regulator of tumor angiogenesis and vascular permeability, and has been implicated both in progression of central nervous system (CNS) tumors and development of vasogenic peritumoral edema. A retrospective study was done to characterize the levels of expression of the 3 major canine VEGF isoforms (VEGF(120), VEGF(164), VEGF(188)) in a variety of spontaneous canine CNS tumors using quantitative TaqMan reverse transcription real-time polymerase chain reaction. Presence and degree of peritumoral edema also were determined in sampled tumors using magnetic resonance imaging (MRI). Increased expression of VEGF relative to normal cerebral cortex tissue was seen predominantly in high grade astrocytic (grade IV) and oligodendroglial (grade III) tumors, with lower expression in low grade astrocytomas (grade II) and meningiomas (grade I). All 3 major VEGF isoforms were present; VEGF(164) was the predominant isoform, particularly in the tumors with the highest VEGF expression. Peritumoral edema was present in all tumor types; however, a significant association between the extent of peritumoral edema and the level of VEGF expression was not apparent.

Endostatin and Vascular Endothelial Growth Factor Concentrations in Healthy Dogs, Dogs with Selected Neoplasia, and Dogs with Nonneoplastic Diseases

To evaluate the relationship between endostatin and vascular endothelial growth factor (VEGF) in cancers of dogs, circulating concentrations of these 2 tumor‐associated markers were measured prospectively in healthy dogs (n = 44), dogs with tumors (n = 54), and dogs with nonneoplastic diseases (n = 42 for endostatin; n = 16 for VEGF). A canine‐directed enzyme‐linked immunosorbent assay kit was used for determination of endostatin, and a human‐directed kit was validated for detection of canine VEGF. Concentrations of endostatin for all dogs were 28–408 ng/mL. Increasing serum endostatin concentration was associated with increasing age (P= .0396). Concentrations of endostatin were not different among groups of dogs (P= .1989) when adjusted for age. Mean endostatin concentrations for all dogs were higher in dogs (P= .0124) with detectable VEGF concentrations. Endostatin concentrations, when corrected for age, were related to decreasing PCV (P= .032) but not white blood cell count (P= .225) or platelet count (P= .1990). Measurable VEGF (≥2.5 pg/mL) was detected in 3 (7.0%) of 43 healthy dogs. Dogs with tumors had detectable VEGF in 24 (44%) of 54 dogs, with concentrations ranging from 2.5–274 pg/mL; only 1 dog with a nonneoplastic disease process had detectable VEGF. VEGF concentrations for all dogs after correcting for age, endostatin, and disease categories were associated with increased white blood cell count (P= .0032) and platelet counts (P= .0064) and decreased PCV (P= .0017). Linkage between increased endostatin and VEGF concentrations suggests that similar factors may influence concentrations of these markers. Further evaluation of endostatin and VEGF associations in dogs with tumors may provide information on the extent and progression of the disease.

Endostatin and Vascular Endothelial Growth Factor Concentrations in Healthy Dogs, Dogs with Selected Neoplasia, and Dogs with Nonneoplastic Diseases

To evaluate the relationship between endostatin and vascular endothelial growth factor (VEGF) in cancers of dogs, circulating concentrations of these 2 tumor-associated markers were measured prospectively in healthy dogs (n = 44), dogs with tumors (n = 54), and dogs with nonneoplastic diseases (n = 42 for endostatin; n = 16 for VEGF). A canine-directed enzyme-linked immunosorbent assay kit was used for determination of endostatin, and a human-directed kit was validated for detection of canine VEGF. Concentrations of endostatin for all dogs were 28-408 ng/mL. Increasing serum endostatin concentration was associated with increasing age (P = .0396). Concentrations of endostatin were not different among groups of dogs (P = .1989) when adjusted for age. Mean endostatin concentrations for all dogs were higher in dogs (P = .0124) with detectable VEGF concentrations. Endostatin concentrations, when corrected for age, were related to decreasing PCV (P = .032) but not white blood cell count (P = .225) or platelet count (P = .1990). Measurable VEGF (> or = 2.5 pg/mL) was detected in 3 (7.0%) of 43 healthy dogs. Dogs with tumors had detectable VEGF in 24 (44%) of 54 dogs, with concentrations ranging from 2.5-274 pg/mL; only 1 dog with a nonneoplastic disease process had detectable VEGF. VEGF concentrations for all dogs after correcting for age, endostatin, and disease categories were associated with increased white blood cell count (P = .0032) and platelet counts (P = .0064) and decreased PCV (P = .0017). Linkage between increased endostatin and VEGF concentrations suggests that similar factors may influence concentrations of these markers. Further evaluation of endostatin and VEGF associations in dogs with tumors may provide information on the extent and progression of the disease.

Serum and Plasma Vascular Endothelial Growth Factor Levels in Various Cat Populations

Introduction: Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Elevated levels within tumors, and in serum, plasma, and tumor effusion have been correlated with the development of metastatic disease, recurrence, and poor prognosis in many tumors in humans. Canine VEGF has been sequenced as homologous with the human form, and elevated serum and plasma VEGF have been found in dogs with hemangiosarcoma. Feline VEGF has also been sequenced, and shares homology with the human and canine forms.Materials and Methods: Stored serum and plasma samples from normal cats, cats with various neoplasms, and cats with non‐neoplastic disease were evaluated with a commercial ELISA kit (R&amp;D Systems, Minneapolis MN). Samples were run in duplicate, and a standard curve was performed for each plate. The data were analyzed for differences between populations, and between serum and plasma levels in the same patient to determine the optimal sample for evaluating VEGF in cats.Results: In seven apparently healthy cats mean plasma VEGF was 95.6 pg/mL. In non‐neoplastic disease (7 cases), mean plasma VEGF was 117.3 pg/mL and mean serum VEGF level was 219.7 pg/mL. In ten tumor‐bearing patients mean plasma VEGF was 247.1 pg/mL, and mean serum VEGF was 322.3 pg/mL. Statistical analysis showed no significant difference between mean serum and plasma VEGF concentrations within each group or between groups (p &gt; 0.05).Discussion: Serum and plasma VEGF levels could not be used to distinguish between healthy cats and cats with neoplastic or non‐neoplastic disease.

Vascular endothelial growth factor (VEGF) and its receptors in tumor-bearing dogs.

The molecular biology of the angiogenic growth factor, vascular endothelial growth factor (VEGF), has been studied in the dog. All major isoforms of VEGF are present in the dog. The amino acid sequences are identical between human and dog in the loop regions that are responsible for receptor binding. Accordingly, the VEGF receptors of dogs and humans are very similar and permit functional exchange of the growth factor. Here we show that canine VEGF activates human endothelial cells to the same extent as human VEGF. Similarly, the two proteins display identical cell binding properties. The VEGF receptor 1 (Flt-1) shows the same alternative splicing in humans and dogs and is overexpressed in the majority of tumors in both species. VEGF occurs also in canine tumors in similar relative quantities as in human malignancies. Based on the literature and our study we suggest that the molecular biology and the function of the VEGF signaling system are virtually identical in humans and canines and in healthy as well as in disease conditions.