Endothelial Cell-derived Growth Factor Research Articles

Abstract 4301: Endothelial angiopoietin-2 expression correlates with tumor angiogenesis and response to sunitinib in metastatic renal cell carcinoma

Abstract Background: Angiopoietin-2 (Ang2) is an endothelial cell derived growth factor, which promotes tumor angiogenesis, tumor growth and metastasis. Ang2 is a promising target for anti-angiogenic therapies, but its potential as a biomarker for anti-angiogenic therapies remains unknown. The purpose of this study was to evaluate expression of the Ang2-Tie2 signalling pathway components in metastatic RCC (mRCC) patients treated with sunitinib, a modern multitarget TKI. Materials and methods: We evaluated Ang2, CD31 and Ki67 expression in tumors of 136 patients with metastatic RCC (mRCC) treated with first-line sunitinib from formalin-fixed paraffin-embedded tumor samples using immunohistochemistry. Results: Ang2 was exclusively expressed in the endothelial cells of tumor blood vessels. Using a cutoff of ≥50 Ang2+ vessels/microscopic field, 38% of the samples had high Ang2 expression. High Ang2 expression correlated with high CD31+ vessel density (p = 0.002). 91% of patients with high Ang2 expression had partial response (PR) or stable disease (SD) as best response to sunitinib as compared with 76% of those with low Ang2 expression (P = 0.033). High CD31+ vessel density correlated with a high clinical benefit rate (P = 0.015), and with low levels of Ki-67+ tumor cells (P = 0.018). Combined high expression of both Ang2 and CD31 predicted even better clinical benefit rate with 100% of the patients with high Ang2 and CD31 expression (n = 25) achieving PR/SD as compared with 77% among the rest of the patients (n = 101; P = 0.007). Only one patient (8%, n = 12) with papillary RCC, but 49 (41%) patients with clear cell RCC had high Ang2 expression (P = 0.03). Conclusions: In this first study to investigate Ang2 expression and outcome in mRCC patients treated with sunitinib, high Ang2 expression in the tumor vasculature was predictive for sunitinib efficacy. Citation Format: Anita Lampinen, Juhana Rautiola, Tuomas Mirtti, Ari Ristimäki, Heikki Joensuu, Petri Bono, Pipsa Saharinen. Endothelial angiopoietin-2 expression correlates with tumor angiogenesis and response to sunitinib in metastatic renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2015-4301

Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment.

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.

Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

194: A molecular profile of endothelial cell-derived growth factors that regulate stem cell self-renewal

Several molecular pathways that regulate hematopoietic stem cell (HSC) self-renewal in the mouse have recently been elucidated, including Notch, HOXB4, Wnt and bone morphogenetic protein signaling pathways. However, full elucidation of the intrinsic and extrinsic mechanisms that regulate human HSC self renewal has yet to be realized. As a model for identifying extrinsic factors that stimulate human HSC self-renewal, we have studied the capacity for vascular endothelial cells to support human HSC expansion ex vivo.

Renal Involvement in Multicentric Castleman Disease With Glomeruloid Hemangioma of Skin and Plasmacytoma

A 47-year-old man presented with fever of unknown origin, generalized weakness, edema, and renal failure. He had left-sided pleural effusion, generalized lymphadenopathy, multiple nontender cutaneous nodules, hepatomegaly, renal failure, and hypergammaglobulinemia. Axillary lymph node biopsy showed findings consistent with Castleman disease of the hyaline vascular type associated with interfollicular plasmacytosis. A renal biopsy performed in view of proteinuria and acute renal failure showed hypercellular glomeruli with capillary loop thickening and double contours consistent with membranoproliferative glomerulonephritis. Skin nodule biopsy showed a glomeruloid hemangioma characterized by dermal proliferation of capillary loops in a nodular manner resembling a glomerulus. He experienced clinical and biochemical remission with steroids. Discontinuation of steroid therapy was associated with recurrence of renal failure, reappearance of nodules, lymphadenopathy, and appearance of bony lytic lesions. Biopsy of bony lytic lesions showed plasmacytoma. The patient achieved complete remission on treatment with steroids and cyclophosphamide and is free of symptoms at the end of 40 months of follow-up. To our knowledge, this is the first case report of the occurrence of membranoproliferative glomerulonephritis, glomeruloid hemangioma of the skin, and plasmacytoma in a patient with multicentric Castleman disease without human immunodeficiency virus infection.

Endothelial Cells Mediate the Repair of Human Bone Marrow Stem and Progenitor Cells Following Radiation Injury.

The purposeful misuse of ionizing radiation has been recognized as a major bioterrorism threat in the United States. Although the myeloablative effects of ionizing radiation exposure have been well established, definitive treatments for potential victims of radiation injury are lacking. We have recently demonstrated that BM stem cells can be harvested from mice following exposure to lethal dose total body irradiation and can subsequently recover multilineage repopulating potential via ex vivo culture with microvascular endothelial cells (EC). However, it remains to be determined whether human hematopoietic stem cells (HSC) can be recovered in a similar manner following high dose radiation injury. In this study, we examined the capacity for ex vivo culture with and without primary human endothelial cells to support the cellular repair and recovery of primary human BM CD34+ cells following exposure to high dose irradiation. Exposure of primary BM CD34+ cells to 400 cGy resulted in a 40% decline in total viable cells and an 89% decline in CD34+CD38− cells compared to input despite 10 day culture with optimal concentrations of thrombopoietin, stem cell factor, and flt-3 ligand alone. Conversely, co-culture of 400 cGy irradiated BM CD34+ cells with primary human brain endothelial cells (LS-01) resulted in a 6-fold and 22-fold increase in total cells and CD34+CD38− cells compared to input, respectively. Non-contact cultures with LS-01 resulted in comparable recovery of total cells and the CD34+CD38− subset by 10 days. Concordantly, TSF-cultured progeny contained 5-fold greater numbers of early apoptotic and necrotic cells within the total and CD34+CD38− fractions as compared to the progeny of contact and non-contact endothelial cultures. Colony forming cell (CFC) assays demonstrated that 400 cGy exposure caused an 8-fold reduction in CFU-total compared to normal BM CD34+ cells and no recovery of CFU-GM, BFU-E, or CFU-Mix was observed following TSF culture. Co-culture with LS-01 resulted in the recovery of 50% of CFU-total compared to normal BM CD34+ cells. In order to assess HSC content post-irradiation, NOD/SCID mice were transplanted with normal BM CD34+ cells, 400 cGy irradiated BM CD34+ cells, and the progeny of 400 cGy irradiated cells following ex vivo culture. 100% of mice transplanted with normal BM CD34+ cells (7.5 x 105) demonstrated human hematopoietic engraftment at 6 weeks, whereas 0% of mice transplanted with 400 cGy irradiated BM CD34+ cells showed detectable human repopulation. Twenty-five percent of mice transplanted with 400 cGy irradiated/LS-01 cultured cells demonstrated human repopulation, whereas 0% of mice transplanted with 400 cGy irradiated/TSF-cultured cells showed human engraftment. These data demonstrate that ionizing radiation has a profoundly toxic effect on both human HSC and committed progenitor cells. Endothelial cells and endothelial cell-derived soluble factors appear to provide anti-apoptotic signals to BM progenitor cells following radiation damage, allowing cellular repair of committed progenitors as well as cells with in vivo repopulating capacity. Cytokine combinations alone, such as TSF, appear ineffective toward rescuing human BM stem/progenitor cells from radiation damage. Studies are ongoing to optimize the application of endothelial cells and endothelial cell-derived growth factors to stimulate HSC repair following radiation injury.

Thioredoxin Reductase Regulates Angiogenesis by Increasing Endothelial Cell-Derived Vascular Endothelial Growth Factor

: Low selenium (Se) status increases angiogenesis by inducing the production of vascular endothelial growth factor (VEGF); however, the mechanism responsible for VEGF up-regulation has yet to be characterized. Se's ability to control cellular oxidative state through its incorporation into selenoproteins such as thioredoxin reductase (TrxR) may explain previous studies that connect Se status to tumor angiogenesis. Therefore, the focus of this study was to determine if altered VEGF expression and angiogenesis due to decreased Se levels are influenced by reduced TrxR activity. We found that chemical inhibition of TrxR in Se-sufficient endothelial cells (ECs) was associated with increases in VEGF and VEGF receptor expression, cell migration, proliferation, and angiogenesis to levels similar to those seen in Se-deficient ECs. Specific inhibition of glutathione peroxidase did not affect pro-angiogenic responses, indicating a unique role of the TrxR system during low Se status. These data correlate changes in TrxR activity with changes in VEGF expression and angiogenic development in ECs, which is significant because minimal mechanistic data exist that explain the role of Se in cancer prevention. Understanding the importance of the tumor microenvironment in contributing to angiogenic regulation has the potential to significantly impact breast cancer chemoprevention strategies by focusing on maintaining proper EC function within the mammary gland.

Multicentric Castleman's disease associated with glomerular microangiopathy and MPGN-like lesion: does vascular endothelial cell-derived growth factor play causative or protective roles in renal injury?

A 52-year-old Japanese man presented with fever spikes, generalized fatigue, anorexia, and anasarca. The patient was referred for the evaluation of fever of unknown origin in association with swelling of cervical, axillary, and inguinal lymph nodes. He also manifested nephrotic syndrome, acute renal failure, hepatosplenomegaly, massive pleural effusion, ascites, disseminated intravascular coagulation, and hypergammaglobulinemia. C-reactive protein was positive and plasma vascular endothelial cell-derived growth factor (VEGF) and serum interleukin-6 levels were markedly elevated. Lymph node biopsy results showed that findings were compatible with Castleman's disease of hyaline vascular type associated with interfollicular plasmacytosis. In conjunction with the clinical findings, a diagnosis of multicentric Castleman's disease was made. The patient underwent renal biopsy because of nephrotic syndrome, and the results showed proliferation of mesangial cells, lobulation of glomeruli, and tram track pattern of the capillary wall without immune complex deposition. Electron microscopy showed widening of the subendothelial space. No electron-dense deposits were present in both mesangial and subendothelial regions. Pathologic features were compatible with glomerular microangiopathy and membranoproliferative glomerulonephritis-like lesions. With corticosteroid therapy, systemic symptoms disappeared; both VEGF and interleukin-6 levels were normalized, and he went into complete remission of nephrotic syndrome. In this article, the role VEGF plays in the pathogenesis of nephrotic syndrome and glomerular microangiopathy is discussed.

The effect of the LysoPC-induced endothelial cell conditioned medium on proliferating cell nuclear antigen expression of the calf thoracic aorta smooth muscle cells.

In order to study the effect of and mechanism of lysophosphatidylcholine (LysoPC) on proliferation of the calf thoracic aorta smooth muscle cells (ASMCs), the ASMCs were used to observe the effects of LysoPC-induced endothelial cell conditioned medium on the DNA content and proliferating cell nuclear antigen (PCNA) expression in the calf thoracic ASMCs by flow cytometry and Western Blot technique. It was found that LysoPC-induced endothelial cell conditioned medium could significantly promote PCNA expression of the calf ASMCs, induce the converting of ASMCs from G0/G1 phase to S phase of DNA synthesis, and increase the tyrosine phosphorylation protein expression. Tyrosine protein kinase inhibitor (TPKi) RG50864 could obviously inhibit proliferation of LysoPC-induced ASMCs in a dose-dependence manner. The results indicated that the effect of LysoPC promoting the proliferation of ASMCs is partly evoked by endothelial cell derived growth factors such as PDGF and so on.

Ocular Neovascularization: Clarifying Complex Interactions

Neovascularization within the eye contributes to visual loss in several ocular diseases, the most common of which are proliferative diabetic retinopathy, neovascular age-related macular degeneration, and retinopathy of prematurity. Together, these three diseases afflict persons in all stages of life from birth through late adulthood and account for most instances of legal blindness. Retinopathy of prematurity (ROP) occurs in premature neonates. Normally, the retina becomes completely vascularized at full term. In the premature baby, the retina remains incompletely vascularized at the time of birth. Rather than continuing in a normal fashion, vasculogenesis in the premature neonatal retina becomes disrupted. Abnormal new proliferating vessels develop at the juncture of vascularized and avascular retina. These abnormal new vessels grow from the retina into the vitreous, resulting in hemorrhage and tractional detachment of the retina. Although laser ablation of avascular peripheral retina may halt the neovascular process if delivered in a timely and sufficient manner, some premature babies nevertheless go on to develop retinal detachment. Surgical methods for treating ROP-related retinal detachments in neonates have limited success at this time because of unique problems associated with this surgery, such as the small size of the eyes and the extremely firm vitreoretinal attachments in neonates. Diabetic retinopathy is the leading cause of blindness in adults of working age. In persons with diabetes mellitus, retinal capillary occlusions develop, creating areas of ischemic retina. Retinal ischemia serves as a stimulus for neovascular proliferations that originate from pre-existing retinal venules at the optic disk or elsewhere in the retina posterior to the equator. Severe visual loss in proliferative diabetic retinopathy (PDR) results from vitreous hemorrhage and tractional retinal detachment. Again, laser treatment (panretinal photocoagulation to ischemic retina) may arrest the progression of neovascular proliferations in this disease but only if delivered in a timely and sufficiently intense manner. Some diabetic patients, either from lack of ophthalmic care or despite adequate laser treatment, go on to sustain severe visual loss secondary to PDR. Vitrectomy surgery can reduce but not eliminate severe visual loss in this disease. Age-related macular degeneration is the leading cause of severe visual loss in persons over 65 years old. In contrast to ROP and PDR, in which neovascularization emanates from the retinal vasculature and extends into the vitreous cavity, AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularization causes severe visual loss in AMD patients because it occurs in the macula, the area of retina responsible for central vision. The stimuli which lead to choroidal neovascularization are not understood. Laser ablation of the choroidal neovascularization may stabilize vision in selected patients. However, only 10% to 15% of patients with neovascular AMD have lesions judged to be appropriate for laser photocoagulation according to current criteria. Retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration are but three of the ocular diseases which can produce visual loss secondary to neovascularization. Others include sickle cell retinopathy, retinal vein occlusion, and certain inflammatory diseases of the eye. These, however, account for a much smaller proportion of visual loss caused by ocular neovascularization. Additional treatments beyond laser photocoagulation and vitrectomy surgery are needed to improve outcomes in these patients. Pharmacological antiangiogenic therapy can potentially assist in prevention of the onset or progression of ocular neovascularization and is a current goal of many research laboratories and pharmaceutical companies.

Guided migration as a novel mechanism of capillary network remodeling is regulated by basic fibroblast growth factor.

To investigate mechanisms of capillary network remodeling, we developed a serum-free angiogenesis in vitro system in three-dimensional fibrin matrices which allows the study of directional growth of endothelial sprouts, anastomosis, and remodeling ('pruning') of the primitive plexus toward more elaborated capillary trees. To follow the movements of living endothelial cells by inverse-fluorescence microscopy, we cocultured unlabeled endothelial cells with endothelial cells labeled with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). We show that elongation and retraction of neighboring capillary sprouts occurs simultaneously, resembling a tug-of-war by which endothelial cells are withdrawn from shortening sprouts to become incorporated in other sprouts nearby. For the first time, we directly demonstrate the long-suspected parallel sliding movement of endothelial cells. We show that cell migration persists within immature capillaries even after sprouts have merged to continuous capillary loops, leading to overlapping growth of opposing sprout tips. As a novel concept of capillary remodeling, we distinguish two types of endothelial cell migration: sprouting and guided migration. Sprouting is the de novo invasion of a matrix by endothelial cells, and guided migration is the locomotion of cells along preexistent capillary-like structures. We show that guided migration leads to remodeling of immature capillary networks and to the retraction of sprouts. We describe a method for quantification of sprouting versus guided migration in DiI-mosaic-labeled capillary networks, and we present evidence that endothelial cell-derived basic fibroblast growth factor serves as a chemotactic signal for other cells to migrate along a preestablished capillary-like structure.

Regulation of human aortic endothelial cell-derived mesenchymal growth factors by allogeneic lymphocytes in vitro. A potential mechanism for cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is thought to be triggered by an alloreactive response to the donor coronary vasculature, resulting in smooth muscle cell proliferation and ultimate occlusion of the donor coronary arteries. To determine whether allogeneic lymphocytes are capable of regulating endothelial-derived smooth muscle cell (SMC) growth factors, human aortic endothelial cells (HAECs) were exposed to allogeneic lymphocytes. The HAEC-lymphocyte co-cultures were assessed for (a) lymphocyte proliferation in response to the allogeneic HAECs; (b) release of soluble factors that stimulate human aortic SMC proliferation; and (c) alteration of HAEC mRNA levels for a panel of known SMC growth factors. Co-culture conditioned medium increased SMC proliferation, compared to medium conditioned by HAECs alone. HAECs exposed to allogeneic lymphocytes increased their expression of mRNA for basic fibroblast growth factor, transforming growth factors alpha and beta, and platelet derived growth factor A and B chains. These results demonstrate that allogeneic lymphocytes are capable of inducing HAECs to increase mRNA levels for several mesenchymal growth factors and to release bioactive products capable of stimulating SMC cell proliferation in vitro. Additionally, the data support the hypothesis that alloreactive lymphocytes can stimulate allogeneic donor endothelial cells to produce growth factors that may contribute to the intimal thickening seen in cardiac allograft vasculopathy.

Analysis of secretion and expression of platelet-derived growth factor in cultured endothelial cells from stroke-prone spontaneously hypertensive rat.

1. We characterized the endothelial cell-derived growth factors of SHRSP and Wistar Kyoto rats (WKY), respectively and found that platelet-derived growth factor (PDGF)-B chain related growth factor constituted a major portion of the mitogenic activity of the conditioned media of endothelial cells from both animals. There were no remarkable qualitative differences between the endothelial cell-derived growth factors of SHRSP and WKY. 2. Northern analysis revealed that the expression of PDGF-B chain was 2-4-fold enhanced in cultured aortic endothelial cells of SHRSP. This enhanced expression of PDGF-B chain, which may be induced under chronic hypertensive conditions, is suggested to contribute to the increase in endothelial cell-derived growth factors reported in this animal.

Effects of endothelial-cell-derived growth factors on cultured astrocytes

To clarify the participation of endothelial-cell-derived growth factors (ECDGFs) in astrocytosis, the effects of endothelial-cell-conditioned medium (ECCM) derived either from normotensive rats or spontaneously hypertensive, stroke-prone rats (SHRSPs) on proliferation of C6 cells of an established rat glioma cell line were bioassayed. The ECCM from both strains stimulated proliferation of astrocytes, but the ECCM from SHRSPs showed a higher mitogenic activity for astrocytes than that from normotensive rats. Growth-promoting activity of the ECCM derived from SHRSPs showed an increase that was linear to the conditioning time. These results seem to indicate that endothelial cells produce and release factors that promote the growth of astrocytes. It seems also probable that chronic hypertension causes an increase in production and release of such ECDGFs that correlated with astrocytic proliferation.

Low Molecular Weight Fibrinogen Degradation Products Stimulate the Release of Growth Factors from Endothelial Cells

Cultured porcine aortic endothelial cells (PAEC) constitutively produce and secrete in their culture medium mitogens collectively called endothelial cell-derived growth factors (EDGFs). Incubation of PAEC with fibrinogen-degradation products (FDPs) obtained by plasmin digestion of highly purified fibrinogen caused an increased release of EDGFs, as assessed by [3H]-thymidine incorporation in 3T3 mouse fibroblasts. The effect was time-dependent and correlated with the degree of fibrinogenolysis. It was accompanied by elongation of the cells. Neither increase in EDGFs release nor cell damage was observed when non-degraded fibrinogen was incubated with endothelial cells. Low molecular weight fibrinogen degradation products (LMWFDPs) (M(r) less than or equal to 10,000), and the higher molecular weight fibrinogen fragments D and E were tested under the same conditions. Only the LMWFDPs caused elongation and damage to PAEC and a marked stimulation (up to 12 fold) of EDGFs release. A low density growth assay revealed that the released EDGFs were mitogenically active on the same PAEC. The activity of the released EDGFs was time and dose dependent on both 3T3 fibroblasts and PAEC, indicating that LMWFDPs caused enhanced release of EDGFs that can act in paracrine and autocrine fashion. This study suggests an additional role for fibrinogenolysis contributing to wound healing, and possibly to atherosclerosis.

High yield purification of melanoma growth stimulatory activity

Tumor cells produce a variety of hormones and growth factors that are associated with modulation of the growth pattern of malignant cells. Hs294T human malignant melanoma cells produce a monolayer mitogen, melanoma growth stimulatory activity (MGSA), that stimulates the growth of Hs294T cultures in serum-free medium. MGSA has been purified to homogeneity from conditioned medium of Hs294T human malignant melanoma cells using acetic acid extraction of the crude conditioned medium followed by three Chromatographie processes, including gel-filtration, heparin-Sepharose, and reverse-phase HPLC. MGSA was eluted from the heparin-Sepharose resin with 0.1–0.3 M NaCl. The binding affinity is similar to that of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) but much less than many endothelial cell-derived growth factors which require significantly higher salt concentrations for elution. These procedures resulted in a final yield of purified MGSA that was significantly greater than yields obtained using previously reported procedures. The homogeneous 16000 and 13000 molecular weight moieties obtained by means of these procedures exhibited similar bioactivities (stimulating a 2- to 3-fold increase in Hs294T cell growth) over a 0.06–6 ng concentration range. This bioactivity was progressively inactivated during storage at −80°C. These results indicate that the combination of heparin-Sepharose chromatography and reverse phase-HPLC provides a more efficient means of purification of MGSA.

Rat heart-derived endothelial and smooth muscle cell cultures: Isolation, cloning and characterization

This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC). Populations of these cells derived from both the macro- and microcirculation were obtained utilizing isolated heart perfusion technique. Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion. Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations. Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent. Upon continual subculture late passage (>P 10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes. Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A. All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered ‘hill and valley’ pattern and positive fluorescence for SMC-specific actin and myosin antibodies. Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin. On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures. Cell growth studies on early (<P 10) and late (>P 10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology. Both EC cloned populations and more notably RHE-parent (>P 10) cultures were capable of significant growth when maintained in limiting serum concentration. Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor. These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites. In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.

Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.

Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, have a molecular weight of about 18,400, cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.

Endothelial cells release a chemoattractant for retinal pigment epithelial cells in vitro.

Though the pathogenesis of choroidal neovascular membranes is uncertain, there is evidence to support a primary dysfunction in the retinal pigment epithelium (RPE). This suggests the possibility that a healthy RPE may provide a physical and/or chemical barrier to subretinal endothelial cell invasion. It has recently been shown that RPE cells in culture produce an inhibitor of neovascularization. Histopathologic evidence suggests that RPE cells tend to surround new blood vessels and contain them. We therefore investigated the possibility that RPE cells are guided toward endothelial cells by chemoattractants. Using a modified Boyden chamber technique, we showed that endothelial cells in culture produce a chemoattractant for RPE cells. The active component is trypsin sensitive, stable at extremes of pH (3 through 10), and nondialyzable (12,000- to 14,000-dalton cutoff). It is partially heat stable but becomes completely heat stable in the presence of 1% sodium dodecyl sulfate. These are all characteristics of the previously described endothelial cell-derived growth factor, suggesting that this mitogen might be the chemoattractant. The ability of RPE cells to be attracted to sites of new blood vessel formation may enhance their potential function as inhibitors of neovascularization.

Comparison of intracellular and extracellular mitogenic activity.

Endothelial cell-derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat-stable and trypsin-sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen-secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56 degrees C) and were not inactivated by trypsin. Similar to platelet-derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.