Marsha J. Merrill

Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor.

Brain tumor-associated cerebral edema arises because tumor capillaries lack normal blood-brain barrier function; vascular permeability factor (VPF, also known as vascular endothelial growth factor, VEGF) is a likely mediator of this phenomenon. Clinically, dexamethasone reduces brain tumor-associated vascular permeability through poorly understood mechanisms. Our goals were to determine if suppression of permeability by dexamethasone might involve inhibition of VPF action or expression, and if dexamethasone effects in this setting are mediated by the glucocorticoid receptor (GR). In two rat models of permeability (peripheral vascular permeability induced by intradermal injection of 9L glioma cell-conditioned medium or purified VPF, and intracerebral vascular permeability induced by implanted 9L glioma), dexamethasone suppressed permeability in a dose-dependent manner. Since 80% of the permeability-inducing activity in 9L-conditioned medium was removed by anti-VPF antibodies, we examined dexamethasone effects of VPF expression in 9L cells. Dexamethasone inhibited FCS- and PDGF-dependent induction of VPF expression. At all levels (intradermal, intracranial, and cell culture), dexamethasone effects were reversed by the GR antagonist mifepristone (RU486). Dexamethasone may decrease brain tumor-associated vascular permeability by two GR-dependent mechanisms: reduction of the response of the vasculature to tumor-derived permeability factors (including VPF), and reduction of VPF expression by tumor cells.

VEGF165 Expressed by a Replication-Deficient Recombinant Adenovirus Vector Induces Angiogenesis In Vivo

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.

Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that also induces vascular permeability and macrophage migration. VEGF expression is weak in normal adult brain, but is strongly upregulated in glioma cells and reactive astrocytes, suggesting that chronic overexpression of VEGF in the brain contributes to blood-brain barrier (BBB) breakdown. We examined the effects of chronic VEGF overexposure on the integrity of the BBB using the following approaches: 1) continuous intracerebral infusion of VEGF via miniosmotic pump; and 2) intracerebral injection of an adenoviral vector encoding the VEGF165 gene (AdCMV.VEGF). After 6 days both treatments produced approximately 10-fold breakdown of the BBB (as measured by transport of 14C-aminoisobutyric acid (AIB) from blood into brain) compared with the respective controls (albumin infusion or AdCMV.beta gal virus). BBB disruption in AdCMV.VEGF-treated brains was accompanied by a severe inflammatory response not observed in brains receiving AdCMV.beta gal or VEGF protein infusion, indicating that neither VEGF nor viral particles alone were responsible for the inflammatory response. However, injection of AdCMV.beta gal followed by VEGF infusion to the same site also elicited inflammation. Chronic overexposure of normal brain to VEGF also increased intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I and II expression. Although VEGF itself is not inflammatory, VEGF may modulate immune responses in the central nervous system (CNS) by opening the BBB, altering the immunoprivileged status of the brain, and allowing contact between normally sequestered CNS antigens and blood-borne immune mediators.

Differential Expression of Vascular Endothelial Growth Factor (Vascular Permeabilty Factor) Forms in Rat Tissues

Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), exists as multiple forms due to alternative splicing of mRNA. VEGF165/164 (human/rodent homologue) is often assumed to be the predominant form, although truly quantitative assessments are lacking. We have used the RNase protection assay to directly quantitate the relative abundance of VEGF mRNA forms in five rat tissues (brain, kidney, lung, spleen, and heart) and two rat glioma cell lines (C6 and 9L). The three major forms, which code for proteins of 188, 164, and 120 amino acids, were observed in all of the tissues and cells examined. However, the relative abundance differed among the samples. VEGF188 was the predominant form (> 50% of total VEGF mRNA) in heart and lung, but was the least abundant form (6-15%) in all other samples. VEGF164 was lower (approximately 25%) in heart and lung, but was predominant (> 50%) in brain and kidney. VEGF164 and VEGF120 were present in equimolar amounts (each form approximately 46% of total) in the spleen, C6, and 9L. VEGF120 was also present in kidney (38%) and lung (27%) and was least abundant (approximately 15%) in brain and heart. A rat homologue of VEGF206 was not observed. VEGF mRNA splicing occurs in a tissue-specific manner. The assumption that the predominant physiologic form of VEGF is a VEGF165/164 homodimer should be viewed with caution.

Real-time image-guided direct convective perfusion of intrinsic brainstem lesions: Technical note

Recent preclinical studies have demonstrated that convection-enhanced delivery (CED) can be used to perfuse the brain and brainstem with therapeutic agents while simultaneously tracking their distribution using coinfusion of a surrogate magnetic resonance (MR) imaging tracer. The authors describe a technique for the successful clinical application of this drug delivery and monitoring paradigm to the brainstem. Two patients with progressive intrinsic brainstem lesions (one with Type 2 Gaucher disease and one with a diffuse pontine glioma) were treated with CED of putative therapeutic agents mixed with Gd-diethylenetriamene pentaacetic acid (DTPA). Both patients underwent frameless stereotactic placement of MR imaging-compatible outer guide-inner infusion cannulae. Using intraoperative MR imaging, accurate cannula placement was confirmed and real-time imaging during infusion clearly demonstrated progressive filling of the targeted region with the drug and Gd-DTPA infusate. Neither patient had clinical or imaging evidence of short- or long-term infusate-related toxicity. Using this technique, CED can be used to safely perfuse targeted regions of diseased brainstem with therapeutic agents. Coinfused imaging surrogate tracers can be used to monitor and control the distribution of therapeutic agents in vivo. Patients with a variety of intrinsic brainstem and other central nervous system disorders may benefit from a similar treatment paradigm.

Vascular endothelial growth factor (vascular permeability factor) expression in injured rat brain

We investigated the expression of vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) in stab and freeze brain injury models in rats. Immunohistochemical staining with anti‐VEGF antibodies demonstrated an increase in VEGF‐positive cells in and around both lesions. Morphologically, the injury‐induced VEGF‐positive cells resembled astrocytes. Double immunofluorescent staining for the astrocytic marker glial fibrillary acidic protein (GFAP) and VEGF demonstrated directly that VEGF‐positive cells which appeared in response to these injuries were astrocytes. VEGF expression in astrocytes was maximal on days 3 and 4 after injury in terms of both cell number and affected area. The increase in VEGF‐positive cells was more widespread in the freeze lesion than in the stab wound, and occurred in both the lesioned and nonlesioned hemispheres. VEGF‐positive cells were still present 3 weeks after both injuries, but their numbers were reduced and their distribution became limited to the immediate vicinity of the lesions. These observations indicate that astrocytes react to injury by increasing VEGF expression, suggesting that VEGF might participate in the central nervous system response to injury. J. Neurosci. Res. 49:451–460, 1997.

Edema is a precursor to central nervous system peritumoral cyst formation.

Despite the common occurrence and frequent clinical effects of peritumoral cysts in the central nervous system (CNS), the mechanism underlying their development and evolution is not understood. Because they commonly produce peritumoral cysts and because serial magnetic resonance imaging (MRI) is obtained in von Hippel–Lindau disease patients, hemangioblastomas provide an opportunity to examine the pathophysiology of CNS peritumoral cyst formation. Serial MRI was correlated with the clinical findings in 16 von Hippel–Lindau disease patients with 22 CNS hemangioblastomas (11 spinal cord; 11 cerebellar) that were associated with the appearance and evolution of peritumoral cysts. Hemangioblastoma‐associated cyst wall histomorphological analysis was performed on postmortem tissues from three von Hippel–Lindau disease patients (not in the clinical series). Comparative proteomic profiling was performed on peritumoral cyst fluid and serum. Vascular endothelial growth factor levels were determined in peritumoral cysts. MRI clearly showed peritumoral edema that developed and slowly and progressively evolved into enlarging hemangioblastoma‐associated cysts in all tumors (mean follow‐up, 130 ± 38 months; mean ± standard deviation). Postcontrast MRI demonstrated convective leakage of gadolinium into cysts. Mean time required for edema to evolve into a cyst was 36 ± 23 months (range, 8–72 months). Thirteen (59%) hemangioblastoma‐cysts became symptomatic (mean time to symptom formation after cyst development, 35 ± 32 months; range, 3–102 months) and required resection. Protein profiles of cyst fluid and serum were similar. Mean cyst fluid vascular endothelial growth factor concentration was 1.5ng/ml (range, 0–5.4ng/ml). Histology of the cyst walls was consistent with reactive gliosis. CNS peritumoral cyst formation is initiated by increased tumor vascular permeability, increased interstitial pressure in the tumor, and plasma extravasation with convective distribution into the surrounding tissue. When the delivery of plasma from the tumor exceeds the capacity of the surrounding tissue to absorb the extravasated fluid, edema (with its associated increased interstitial pressure) and subsequent cyst formation occur. Ann Neurol 2005;58:392–399

Expression of hypoxia-inducible carbonic anhydrases in brain tumors

Malignant brain tumors exhibit distinct metabolic characteristics. Despite high levels of lactate, the intracellular pH of brain tumors is more alkaline than normal brain. Additionally, with increasing malignancy, brain tumors display intratumoral hypoxia. Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes that are induced by tissue hypoxia. They participate in regulation of pH homeostasis by catalyzing the reversible hydration of carbon dioxide. The aim of our study was to investigate whether brain tumors of different histology and grade of malignancy express elevated levels of CA IX and XII as compared to normal brain. We analyzed 120 tissue specimens from brain tumors (primary and metastatic) and normal brain for CA IX and XII expression by immunohistochemistry, Western blot, and in situ hybridization. Whereas normal brain tissue showed minimal levels of CA IX and XII expression, expression in tumors was found to be upregulated with increased level of malignancy. Hemangioblastomas, from patients with von Hippel-Lindau disease, also displayed high levels of CA IX and XII expression. Comparison of CA IX and XII staining with HIF-1alpha staining revealed a similar microanatomical distribution, indicating hypoxia as a major, but not the only, induction factor. The extent of CA IX and XII staining correlated with cell proliferation, as indicated by Ki67 labeling. The results demonstrate that CA IX and XII are upregulated in intrinsic and metastatic brain tumors as compared to normal brain tissue. This may contribute to the management of tumor-specific acid load and provide a therapeutic target.

Function of carbonic anhydrase IX in glioblastoma multiforme.

Carbonic anhydrase (CA) IX is over-expressed in glioblastoma; however, its functions in this context are unknown. Metabolically, glioblastomas are highly glycolytic, leading to a significant lactic acid load. Paradoxically, the intracellular pH is alkaline. We hypothesized that CAIX contributes to the extrusion of hydrogen ions into the extracellular space, thereby moderating intra- and extracellular pH and creating an environment conductive to enhanced invasion. We investigated the role of CAIX as a prognostic marker in patients with glioblastoma and its biological function in vitro. CAIX expression was analyzed in 59 patients with glioblastoma by immunohistochemistry. The expression levels were correlated to overall survival. In vitro, U251 and Ln 18 glioblastoma cells were incubated under hypoxia to induce CAIX expression, and RNA interference (RNAi) was used to examine the function of CAIX on cell attachment, invasion, intracellular energy transfer, and susceptibility to adjuvant treatment. High CAIX expression was identified as an independent factor for poor survival in patients with glioblastoma. In vitro, cell attachment and invasion were strongly reduced after knockdown of CAIX. Finally, the effects of radiation and chemotherapy were strongly augmented after CAIX interference and were accompanied by a higher rate of apoptotic cell death. CAIX is an independent prognostic factor for poor outcome in patients with glioblastoma. Cell attachment, invasion, and survival during adjuvant treatment are significantly influenced by high CAIX expression. These results indicate that inhibition of CAIX is a potential metabolic target for the treatment of patients with glioblastoma.