Kevin P. Corke

Hypoxia-Induced Gene Expression in Human Macrophages: Implications for Ischemic Tissues and Hypoxia-Regulated Gene Therapy

Macrophages accumulate in ischemic areas of such pathological tissues as solid tumors, atherosclerotic plaques and arthritic joints. Studies have suggested that hypoxia alters the phenotype of macrophages in a way that promotes these lesions. However, the genes up-regulated by macrophages in such hypoxic tissues are poorly characterized. Here, we have used cDNA array hybridization to investigate the effects of hypoxia on the mRNAs of 1185 genes in primary human monocyte-derived macrophages. As shown previously in other cell types, mRNA levels for vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1) were up-regulated by hypoxia. However, the mRNAs of other genes were also up-regulated including matrix metalloproteinase-7 (MMP-7), neuromedin B receptor, and the DNA-binding protein inhibitor, Id2. The promoters of GLUT-1 and MMP-7 confer hypoxic inducibility on a reporter gene in RAW 264.7 macrophages, indicating that the hypoxic up-regulation of these mRNAs may occur, at least in part, at the transcriptional level. GLUT-1 and MMP-7 mRNA were also shown to be up-regulated in hypoxic macrophages in vitro by real-time RT-PCR, and these proteins were elevated in hypoxic macrophages in vitro and in hypoxic areas of human breast tumors. The hypoxia up-regulated genes identified could be important for the survival and functioning of macrophages in hypoxic diseased tissues, and their promoters could prove useful in macrophage-delivered gene therapy.

Expression of hypoxia-inducible factor 1alpha by macrophages in the rheumatoid synovium: implications for targeting of therapeutic genes to the inflamed joint.

OBJECTIVE To determine if the rheumatoid synovium is a suitable target for hypoxia-regulated gene therapy. METHODS Sequential sections of wax-embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia-inducible factor 1alpha (HIF-1alpha) and CD68 (a pan-macrophage marker). RESULTS HIF-1alpha was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF-1alpha in OA synovia, and it was absent from all of the healthy synovia. CONCLUSION These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia-inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.

Expression of HIF-1alpha by human macrophages: implications for the use of macrophages in hypoxia-regulated cancer gene therapy.

Large numbers of monocytes extravasate from the blood into human tumours, where they differentiate into macrophages. In both breast and prostate carcinomas, these cells accumulate in areas of low oxygen tension (hypoxia), where they respond to hypoxia with the up‐regulation of one or more hypoxia‐inducible factors (HIFs). These then accumulate in the nucleus and bind to short DNA sequences called hypoxia‐response elements (HREs) near or in such oxygen‐sensitive genes as that encoding the pro‐angiogenic factor vascular endothelial growth factor (VEGF). This stimulates gene expression and could explain why, in part, macrophages express abundant VEGF only in avascular, hypoxic areas of breast carcinomas. It also suggests that macrophages could be used to deliver HRE‐regulated therapeutic genes specifically to hypoxic tumour areas. A recent study suggested that hypoxic macrophages accumulate HIF‐2 rather than HIF‐1, prompting the search for HRE constructs that optimally bind HIF‐2 for use in macrophage‐based gene therapy protocols. However, the present study shows that human macrophages accumulate higher levels of HIF‐1 than HIF‐2 when exposed to tumour‐specific levels of hypoxia in vitro; that macrophages in human tumours express abundant HIF‐1; and that expression from HRE‐driven reporter constructs in the human macrophage‐like cell line MonoMac 6 correlates more closely with HIF‐1 than with HIF‐2 up‐regulation under hypoxia. Taken together, these findings suggest that HIF‐1 may be the major hypoxia‐inducible transcription factor in macrophages and that HIF‐1‐regulated constructs are likely to be effective in macrophage delivery of hypoxia‐regulated gene therapy to human tumours. Copyright

Macrophages promote angiogenesis in human breast tumour spheroids in vivo

An in vivo model has been established to study the role of macrophages in the initiation of angiogenesis by human breast tumour spheroids in vivo. The extent of the angiogenic response induced by T47D spheroids implanted into the dorsal skinfold chamber in nude mice was measured in vivo and compared to that induced by spheroids infiltrated with human macrophages prior to implantation. Our results indicate that the presence of macrophages in spheroids resulted in at least a three-fold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. The angiogenic response measured around the spheroids, 3 days after in vivo implantation, was significantly greater in the spheroids infiltrated with macrophages. The number of vessels increased (macrophages vs no macrophages 34±1.9 vs 26±2.5, P<0.01), were shorter in length (macrophages vs no macrophages 116±4.92 vs 136±6.52, P<0.008) with an increased number of junctions (macrophages vs no macrophages 14±0.93 vs 11±1.25, P<0.025) all parameters indicative of new vessel formation. This is the first study to demonstrate a role for macrophages in the initiation of tumour angiogenesis in vivo.

Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells

Aim: Both hypoxia inducible factor 1 (HIF‐1) and basic fibroblast growth factor (bFGF) play important roles in tumour angiogenesis. This study was designed to clarify the cooperative effect of these two mediators in induction of vascular endothelial cell growth factor (VEGF) release from breast cancer and probe possible mechanisms involved. Methods: Release of VEGF from a breast cancer cell line (T47D) was quantitated by enzyme linked immunosorbent assay (ELISA). Expression of HIF‐1 and ERK was assayed using Western blotting. Transient transfection and dual luciferase reporter assay were used to study HIF‐1 transactivity. Results: The data showed that hypoxia induced the expression of HIF‐1&agr; protein, the transactivity of HIF‐1 and the release of VEGF. bFGF further augmented these hypoxic inductions. The PI3K pathway was required for these processes as demonstrated by application of PI3Kinase inhibitor (LY294002) or mutant construct transfections. In contrast, the MEK1 inhibitor PD98059 showed no effect on either activation of HIF‐1 or VEGF release, which is in agreement with our finding that ERK1/2 was not activated by hypoxia. Under hypoxic conditions, bFGF activated the MEK1/ERK pathway. PD98059 blocked the activation of ERK1/2 and suppressed bFGF‐induced HIF‐1 transactivity, yet the protein expression of HIF‐1&agr; or VEGF release was not affected by PD98059. Conclusion: bFGF augments hypoxia induced VEGF release mainly through the PI3K pathway and partly depending on HIF‐1 activity. Elucidation of this mechanism may provide a new target for anti‐angiogenesis in cancer therapy.

Fibrinogen E fragment selectively disrupts the vasculature and inhibits the growth of tumours in a syngeneic murine model.

We recently demonstrated that a fragment of human fibrinogen, fibrinogen E fragment, inhibits the migration and differentiation of human endothelial cells in vitro. Here we show that it exerts similar effects on murine endothelial cells in vitro, and selectively disrupts tumour endothelium in vivo, causing widespread intravascular thrombosis and retarding the growth of CT26 tumours in a syngeneic murine model.

Distribution of TAM relative to blood vessels in prostate carcinoma: correlation with localization of HIF-1α and HIF-2α

Introduction Prostate cancer is the most common form of cancer in men. It is the second leading cause of cancer death in men in the UK and USA after lung cancer. In a variety of tumors, including breast, ovarian, colorectal, and gastric carcinomas, the neoplastic cell population is often outnumbered by CD68-positive tumor-associated macrophages (TAM). TAM are a form of macrophages with an immature phenotype, that are characterized by low expression of carboxypeptidase M and tumor necrosis factor-&agr; and high expression of interleukin (IL)-1 and IL-6, thereby reducing their tumoricidial activities. It also appears that exposure to hypoxia in poorly vascularized areas of tumors may stimulate TAM to release cytokines and enzymes that drive tumor growth, invasion, angiogenesis, and metastasis. Although prostate carcinomas contain significant areas of hypoxia and large numbers of TAM, to date, neither the distribution of TAM relative to blood vessels (and thus hypoxia) nor their expression of hypoxia-inducible factors (HIFs) 1 and 2 has been examined in such tumors. Study design Forty patients with prostate adenocarcinoma were included this study. The sections were double immunostained for the vascular endothelial marker factor VIII, the pan-macrophage marker CD68, and HIF-1&agr; and HIF-2&agr;. The three most vascular areas (vascular ‘hotspots’) were assessed for mean microvessel density using a 25-point Chalkley array graticule at ×250 magnification (field area 0.189 mm2). Thereafter, they were assessed for macrophage density. Macrophage and microvessel densities were similarly assessed in the three areas of highest macrophage density (macrophage ‘hotspots’). The same method was applied for the analysis of HIF-1&agr; and HIF-2&agr; proteins as well as CD68-positive macrophages. Results This study shows that macrophages are prominent in prostate carcinoma and accumulate in hypoxic areas of tissues as there is a significant and reciprocal relationship between angiogenesis and macrophage infiltration in prostate cancer. HIF-1&agr; and HIF-2&agr; were shown to be expressed in small subsets of macrophages, with a higher expression of HIFs in avascular (35 and 40%) areas when compared with vascular areas (18 and 21%). Conclusion The study gives a valuable indication that TAMs can be used to deliver hypoxically-activated therapeutic DNA constructs to prostate tumors. However, further studies are required to detect the expression of other hypoxia-induced transcription factors in macrophages, which might play a role in activating gene expression in hypoxic tumor areas.