Patricia Rockwell

Selective Binding of VEGF to One of the Three Vascular Endothelial Growth Factor Receptors of Vascular Endothelial Cells

VEGF and VEGF are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF a heparin binding capability which VEGF lacks. I-VEGF bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while I-VEGF bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of I-VEGF to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF and VEGF inhibited the binding of I-VEGF to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF by 2-3-fold. These results contrast with previous observations which have indicated that the binding of I-VEGF to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF is susceptible to oxidative damage caused by oxidants such as HO or chloramine-T. VEGF was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF but not the receptor binding ability of damaged VEGF. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF may enable the restoration of damaged VEGF function in processes such as inflammation or wound healing.

A mutagenic screening of various herbs, spices, and food additives

Abstract Chloroform and methanol extracts of various herbs and spices as well as food additives were screened for mutagenicity using the Salmonella/microsome assay of Ames and the Salmonella typhimurium strains TA100 and TA98. The results of this general screening, however, did not provide sufficient information to fully assess the mutagenic potential of certain herbs and spices since the assay of their respective extracts was accompanied by a growth inhibition of the bacterial tester strain. These findings were attributed to the effects of toxic compounds that were presumably contained within the complex mixtures that comprise both herbs and spices. An apparent reduction in the effects of toxicity was observed when separation methods were used as a means to obtain fewer compounds in each of the samples assayed for mutagenicity. Following a separation by column chromatography of the chloroform and methanol extracts of cumin, a dose related response of weak mutagenicity was demonstrated toward TA100 but no...

Vascular endothelial growth factor receptor 2 (VEGFR2) expression in squamous cell carcinomas of the head and neck

Objectives Vascular endothelial growth factor receptor 2 (VEGFR2; Flk‐1 [fetal liver kinase]/KDR [kinase insert domain containing receptor]) has been identified as a high affinity receptor for vascular endothelial growth factor (VEGF) on vascular endothelium. Head and neck squamous cell carcinomas (HNSCC) have already been shown to produce substantial amounts of VEGF. VEGFR2 is supposed to play a major role in tumor‐neoangiogenesis.

Neuroinflammation and J2 prostaglandins: linking impairment of the ubiquitin-proteasome pathway and mitochondria to neurodegeneration

The immune response of the CNS is a defense mechanism activated upon injury to initiate repair mechanisms while chronic over-activation of the CNS immune system (termed neuroinflammation) may exacerbate injury. The latter is implicated in a variety of neurological and neurodegenerative disorders such as Alzheimer and Parkinson diseases, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, HIV dementia, and prion diseases. Cyclooxygenases (COX-1 and COX-2), which are key enzymes in the conversion of arachidonic acid into bioactive prostanoids, play a central role in the inflammatory cascade. J2 prostaglandins are endogenous toxic products of cyclooxygenases, and because their levels are significantly increased upon brain injury, they are actively involved in neuronal dysfunction induced by pro-inflammatory stimuli. In this review, we highlight the mechanisms by which J2 prostaglandins (1) exert their actions, (2) potentially contribute to the transition from acute to chronic inflammation and to the spreading of neuropathology, (3) disturb the ubiquitin-proteasome pathway and mitochondrial function, and (4) contribute to neurodegenerative disorders such as Alzheimer and Parkinson diseases, and amyotrophic lateral sclerosis, as well as stroke, traumatic brain injury (TBI), and demyelination in Krabbe disease. We conclude by discussing the therapeutic potential of targeting the J2 prostaglandin pathway to prevent/delay neurodegeneration associated with neuroinflammation. In this context, we suggest a shift from the traditional view that cyclooxygenases are the most appropriate targets to treat neuroinflammation, to the notion that J2 prostaglandin pathways and other neurotoxic prostaglandins downstream from cyclooxygenases, would offer significant benefits as more effective therapeutic targets to treat chronic neurodegenerative diseases, while minimizing adverse side effects.

HIFα expression in VHL-deficient renal cancer cells is dependent on phospholipase D

Loss of the von Hippel-Lindau (VHL) tumor suppressor gene contributes to proliferative disorders including renal cell carcinoma. The consequence of VHL loss is increased levels of hypoxia-inducible factor-α (HIFα), which is targeted for proteolytic degradation by the VHL gene product pVHL. HIF is a transcription factor that increases the expression of factors critical for tumorigenesis in renal cell carcinoma. We report here another regulatory component of HIFα expression in renal cancer cells. Phospholipase D (PLD), which is commonly elevated in renal and other cancers, is required for elevated levels of both HIF1α and HIF2α in VHL-deficient renal cancer cells. The induction of both HIF1α and HIF2α by hypoxic mimetic conditions was also dependent on PLD in renal cancer cells with restored pVHL expression. The effect of PLD activity upon HIFα expression was at the level of translation. PLD activity also provides a survival signal that suppresses apoptosis induced by serum deprivation in the renal cancer cells. Suppression of HIF2α has been shown to reverse tumorigenesis with renal cancer cells. The finding here that HIF2α expression is dependent on PLD in renal cancer cells suggests that targeting PLD signals may represent an alternative therapeutic strategy for targeting HIF2α in renal cancers where HIF2α is critical for tumorigenesis and elevated PLD activity is common.

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death.

Myc stabilization in response to estrogen and phospholipase D in MCF-7 breast cancer cells.

Estrogen, which has been strongly implicated in breast cancer, suppresses apoptosis in estrogen receptor (ER) positive MCF‐7 breast cancer cells. Phospholipase D (PLD), which is commonly elevated in ER negative breast cancer cells, also suppresses apoptosis. Survival signals generated by both estrogen and PLD are dependent upon elevated Myc expression. We report here that estrogen‐ and PLD‐induced increases in Myc expression are due to reduced turnover of Myc protein. Estrogen and PLD suppressed phosphorylation of Myc at Thr58 – a site that targets Myc for degradation by the proteasome. The data provide a mechanism for elevated Myc expression in hormone‐dependent and hormone‐independent breast cancer.

p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells

Evidence suggests that vascular endothelial growth factor (VEGF) mediates neuroprotection to prevent an apoptotic cell death. The p38 mitogen-activated protein kinase (MAPK) pathway is implicated as an important mediator of neuronal apoptosis but its role in VEGF-mediated neuroprotection is unclear. Herein, we show that treatments with the p38 MAPK inhibitor, SB202190, enhanced VEGF-mediated survival in serum deprived SK-N-SH neuroblastoma cells by decreasing caspase-3/7 activation while increasing the phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and Akt signaled through the VEGF receptor, VEGFR2. A blockade of VEGFR2 signaling with a selective inhibitor, SU1498 or gene silencing with VEGFR2 siRNA in SB202190 treated cells abrogated this prosurvival response and induced high activation levels of caspase-3/7. These findings suggested that the protection elicited by p38 MAPK inhibition in serum starved cells was dependent on a functional VEGF/VEGFR2 pathway. However, p38 MAPK inhibition attenuated caspase-3 cleavage in SU1498/SB202190 treated cells, indicating that p38 MAPK and caspase-3 only contributed in part to the total levels of caspase-3/7 induced by VEGFR2 inhibition. Pretreatments with the pan caspase inhibitor, z-VAD-fmk, prevented the apoptosis induced by VEGFR2 inhibition and promoted survival in serum starved cells irrespective of p38 MAPK inhibition. Collectively, our findings suggest that p38 MAPK exerts a negative effect on VEGF-mediated signaling through VEGFR2 in serum starved neuroblastoma cells. Furthermore, VEGF signals protection against a caspase-mediated cell death that is regulated by p38 MAPK-dependent and -independent mechanisms.

Signaling through the vascular endothelial growth factor receptor VEGFR-2 protects hippocampal neurons from mitochondrial dysfunction and oxidative stress

Vascular endothelial growth factor VEGF (VEGF-A or VEGF₁₆₅) is a potent angiogenic factor that also signals neuroprotection through activation of its cognate receptor VEGFR-2. In this capacity, VEGF signaling can rescue neurons from the damage induced by stressful stimuli many of which elicit oxidative stress. However, the regulatory role that VEGFR-2 plays in providing neuroprotection remains elusive. Therefore, we investigated the effects of VEGFR-2 inhibition on primary cultures of mature hippocampal neurons undergoing nutritional stress. We found that neurons cultured under nutritional stress had increased expression of VEGF and its receptors, VEGFR-1, VEGFR-2, and NP-1, as well as enhanced levels of VEGFR-2 phosphorylation. These neurons also showed increased activation of the prosurvival pathways for MEK/ERK1/2 and PI3K/Akt, enhanced phosphorylation (inactivation) of the proapoptotic BAD, and higher levels of the antiapoptotic protein Bcl-xL, all of which were augmented by treatments with exogenous VEGF and blocked by VEGFR-2 inhibition. The blockade of VEGFR-2 function also elicited a cytotoxicity that was accompanied by caspase-3 activation, induction of hemeoxygenase-1 (HO-1), oxidative stress, and a collapse in the mitochondrial membrane potential (ΔΨ(m)). Knockdown of VEGFR-2 by siRNA generated a similar pattern of redox change and mitochondrial impairment. Pretreatments with VEGF, VEGF-B, or the antioxidant N-acetylcysteine (NAC) rescued SU1498 or siRNA-treated neurons from the mitochondrial dysfunction and oxidative stress induced by VEGFR-2 inhibition in a timely fashion. These findings suggested that VEGF or VEGF-B can provide neuroprotection by signaling through an alternate VEGF receptor. Together, our findings suggest that VEGF signaling through VEGFR-2 plays a critical regulatory role in protecting stressed hippocampal neurons from the damaging effects of an oxidative insult. These findings also implicate VEGFR-1 or NP-1 as compensatory receptors that mediate neuroprotection when VEGFR-2 function is blocked.

A protein-bound form of thioacetamide in liver nucleoli

The cellular distribution of 35S from 35S- thioacetamide was determined in rabbit liver subcellular fractions following its in vivo administration. Of the various fractions isolated, only the nucleolar fraction contained 35S counts that were insoluble in 10% trichloroacetic acid but soluble in trichloroacetic acid if the fraction was treated with trypsin but not RNase or DNase. These results demonstrate that a protein bound form of thioacetamide is present in the nucleolus following in vivo administration of this drug.