Effects of 10-hydroxycamptothecin, delivered from locally injectable poly(lactide-co-glycolide) microspheres, in a murine human oral squamous cell carcinoma regression model.

This study investigated whether local delivery of 10-hydroxycamptothecin provides effective inductive chemotherapy as assessed by significant tumor reduction. Established tumorigenic human oral squamous cell carcinoma cells were used for these experiments. The experimental groups were comprised of: control (blank (no drug) poly(lactide-co-glycolide) (PLGA) microspheres), intraperitoneal 10-hydroxycamptothecin delivery + blank microspheres, local bolus 10-hydroxycamptothecin + blank microspheres, and PLGA controlled-release microspheres. The 10-hydroxycamptothecin dose administered was 12 mg/kg (bolus-intraperitoneal, local) or controlled-release over 10 days. Regardless of delivery route, 10-hydroxycamptothecin significantly reduces tumor volume. However, PLGA microspheres provide significantly higher intratumor-drug concentrations (approximately 10 and 100 fold higher) relative to local bolus and intraperitoneal routes, respectively. Also, only the PLGA microspheres significantly reduced tumor weights. Camptothecin clinical applications are limited by drug inactivation at physiological pH and the need for sustained infusions. However, due to their acidic, camptothecin-stabilizing microclimate, PLGA microspheres could provide a novel delivery system for camptothecin-based induction chemotherapy.

Controlled-release of doxorubicin from poly(lactide-co-glycolide) microspheres significantly enhances cytotoxicity against cultured AIDS-related Kaposi's sarcoma cells.

Subsequent to the introduction of highly active antiretroviral therapy (HAART), there has been a reduction in HIV viral titers and a concomitant decrease in AIDS-related Kaposi's sarcoma. However, as failure rates of HAART approach 30%, concerns arise regarding resurgence in AIDS-KS. Current AIDS-KS therapies fail to provide sustained remissions and yet also result in significant morbidity. Although partially effective, systemic chemotherapy is particularly debilitating to AIDS patients. In this report, we examined the co-incubation of AIDS-KS cells with doxorubicin which was slowly delivered from biodegradable, locally injectable, controlled-release poly(lactide-co-glycolide) (PLGA) microspheres. Local drug delivery systems such as PLGA microspheres can sustain therapeutic intralesional concentrations while minimizing deleterious systemic side effects, providing a pharmacologic advantage at the treatment site. Our data show that controlled release from PLGA microspheres augments doxorubicin cytotoxicity towards AIDS-KS cells without increasing toxicity in nonlesional cells from the AIDS-KS donors. Electron microscopic analysis revealed that PLGA microspheres possess a strong affinity for cell membranes, facilitating doxorubicin delivery to redox-sensitive cell membrane sites. Consistent with their speculated endothelial cell lineage, some of the AIDS-KS cells appeared to engulf microspheres via phagocytosis. Our results suggest that PLGA controlled-release doxorubicin microspheres have potential clinical applicability in management of AIDS-KS.

Sustained angiogenesis enables in vivo transplantation of mucocutaneous derived AIDS-related Kaposi's sarcoma cells in murine hosts.

AIDS-related Kaposi's sarcoma (AIDS-KS), the most prevalent HIV-associated malignancy, is a debilitating, potentially fatal disease. Currently, there is a need for development of AIDS-KS therapies that are not only well tolerated, but also capable of providing sustained remission. Preclinical assessment of pharmacological parameters and therapeutic efficacies are dependent upon in vivo parameters. However, there are currently no animal KS models and mucocutaneous KS cell isolates have proved to be non-tumorigenic in animal hosts. This report describes the development of a murine model that enables in vivo transplantation of 'native' low population doubling level AIDS-KS cells from biopsy-confirmed mucocutaneous lesions. The angiogenic phenotype of in situ AIDS-KS lesions is reconstituted via controlled release of a complete angiogenic peptide, recombinant human basic fibroblast growth factor (bFGF), from locally injectable, biodegradable polylactide-co-glycolide implants. Consequential to the sustained local release of bioactive bFGF, a murine vascular network is established, which facilitates the in vivo transplantation of AIDS-KS cells. Desirable aspects of this model include: low cost murine species, transplantation of non-selected patient cells and use of animal hosts that are T cell-deficient. The transplanted human AIDS-KS cells and extensive murine vascular network create lesions that retain a striking resemblance, at both the gross and microscopic levels, to in situ AIDS-KS tumors. Because the bFGF-induced murine vascular network is analogous to the abundant vascularity present in AIDS-KS lesions, this murine model should provide an excellent vehicle for numerous clinically relevant studies, such as assessment of drug clearance at AIDS-KS lesional sites. Finally, applicability of this method is not restricted to AIDS-related malignancies. Establishment and maintenance of an extensive host vascular network should augment success rates for in vivo transplantation of numerous other human cell strains or lines.

Stabilization of vinca alkaloids encapsulated in poly(lactide-co-glycolide) microspheres.

PURPOSE: The purpose of this study was to stabilize the vinca alkaloids, vincristine sulfate (VCR) and vinblastine sulfate (VBL), in poly(lactide-co-glycolide) (PLGA) microspheres and to release the drugs in a sustained manner for more than a month. METHODS: An oil-in-oil emulsion-solvent extraction method was used to encapsulate VCR and VBL in PLGA50/50 microspheres. Stability and release kinetics of the drugs during the incubation at 37 degrees C in PBS/Tween 80 were assessed by HPLC. Degradation products were identified with HPLC-MS. RESULTS: VCR and VBL were encapsulated in PLGA microspheres unchanged. During the microsphere incubation, however, VCR degraded inside the particles with a t1/2 approximately 7.5 days. The degradation product was identified by LC-MS as the deformyl derivative, commonly formed at acidic pH. VBL, which differs only by a stable methyl group in place of the N-formyl group in VCR, was completely stable in the PLGA microclimate. The neutralization of acidic PLGA microclimate by addition of 3-10% Mg(OH)2 completely inhibited deformylation of VCR during release. but introduced a new degradation product formed under the more alkaline conditions used during the preparation. The substitution of Mg(OH)2 with a weaker base, ZnCO3, inhibited the formation of both degradation products resulting in VCR stabilization of >92% for 4 weeks. The optimal formulations of VCR (containing ZnCO3) and VBL (no additives) slowly and continuously released stable drugs for over a month. CONCLUSIONS: VCR and VBL were successfully stabilized and released in a sustained manner from PLGA microspheres. Co-encapsulation of ZnCO3 stabilizes VCR against acid-catalyzed degradation during release from the polymer and minimizes VCR decomposition during encapsulation.

Stabilization of proteins encapsulated in injectable poly (lactide- co-glycolide)

Controlled release from biodegradable polymers is a novel approach to replace daily painful injections of protein drugs. A major obstacle to development of these polymers is the need to retain the structure and biological activity of encapsulated proteins during months of incubation under physiological conditions. We encapsulated bovine serum albumin (BSA) in injectable poly(DL-lactide- co-glycolide) (PLGA) 50/50 cylindrical implants and determined the mechanism of BSA instability. Simulations of the polymer microclimate revealed that moisture and acidic pH (<3) triggered unfolding of encapsulated BSA, resulting in peptide bond hydrolysis and noncovalent aggregation. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polyester, we coincorporated into the polymer an antacid, Mg(OH)2, which increased microclimate pH and prevented BSA structural losses and aggregation for over one month. We successfully applied this stabilization approach in both cylinder- and microsphere-injectable configurations and for delivery of angiogenic basic fibroblast growth factor and bone-regenerating bone morphogenetic protein-2.

Thiol redox modulation of doxorubicin mediated cytotoxicity in cultured AIDS-related Kaposi's sarcoma cells.

The chemotherapeutic, doxorubicin, is currently used empirically in the treatment of AIDS- related Kaposi's sarcoma (AIDS-KS). Although often employed in a chemotherapeutic cocktail (doxorubicin, bleomycin, vincristine) single-agent therapy has recently been attempted with liposome encapsulated doxorubicin. Although doxorubicin's mechanism of action against AIDS-KS is unknown, we hypothesized that doxorubicin's ability to undergo redox cycling is associated with its clinical efficacy. The current study was conducted to investigate the effects of doxorubicin on selected xenobiotic-associated biochemical responses of three cellular populations: KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV- aged matched men. Our results show that during doxorubicin challenge, there are strong positive correlations between cellular glutathione (GSH) levels and viability (r = 0.94), NADPH levels and viability (r = 0.93), and GSH and NADPH levels (r = 0.93), and demonstrate that as a consequence of their abilities to maintain cellular thiol redox pools HIV- donor cells are significantly less susceptible to doxorubicin's cytotoxic effects relative to AIDS-KS cells. Additional studies further supported the contribution of reduced thiols in mediating doxorubicin tolerance. While pretreatment with the GSH precursor, N-acetylcysteine was cytoprotective for all cell groups during doxorubicin challenge, GSH depletion markedly enhanced doxorubicin's cytotoxic effects. Studies to investigate the effects of a hydroxyl scavenger and iron chelator during doxorubicin challenge showed moderate cytoprotection in the AIDS-KS cells but deleterious effects in the HIV control cells. Inactivation of the longer lived membrane generated ROI in the cytoprotective deficient AIDS-KS cells, as well as an impairment of endogenous defenses in the HIV- donor control cells, may account for these scavenger and chelator associated findings. In summary, our findings show that doxorubicin mediates, at least in part, its AIDS-KS cellular cytotoxic effects by a redox related mechanism, and provides a biochemical rationale for doxorubicin's clinical efficacy in AIDS-KS treatment.

The endogenous antioxidant glutathione as a factor in the survival of physically injured mammalian spinal cord neurons.

Glutathione is part of the system of cellular defenses against lipid peroxidation and other free radical-mediated damage. An established in vitro trauma model was utilized to evaluate whether glutathione is a factor in the survival of mammalian spinal cord neurons following physical injury. Cultured murine spinal neurons were subjected to a standard lesion: transection of a primary dendrite 100 microm from the perikaryon. Prior reduction of glutathione with ethacrynic acid or buthionine sulfoximine caused a dose-dependent decrease in neuronal survival 24 hours after dendrotomy. Prior glutathione augmentation with gamma-glutamylcysteine or L-2-oxo-4-thiazolidine carboxylic acid significantly increased survival, but N-acetyl-cysteine was not protective. Gamma glutamylcysteine effected the most rapid increase in glutathione (peak at 10 min), and survival was 72% +/- 10 when 0.2 mM gamma-glutamylcysteine was added immediately after dendrotomy compared with 38% +/- 4 in the control group (p < 0.0001). These results indicate that the level of glutathione is a factor in spinal cord neuron survival after physical trauma, and that glutathione augmentation may be an effective acute phase spinal cord injury (SCI) intervention strategy.

Stress-related modulation of central nervous system immunity in a murine model of herpes simplex encephalitis.

This study assesses the immunomodulatory effects of stress on the pathogenesis of herpes simplex encephalitis (HSE) in a mouse model. Physical restraint served as the stressor and HSE developed subsequent to HSV-1 inoculation into the tongues of subject animals. Clinical data showed that stressed mice lost more weight and had greater mortality rates than unrestrained animals during the course of infection. Histologic tissue sections demonstrated a stress-related reduction of the cellular inflammatory response in the central nervous system (CNS). This model may be useful to further investigate the mechanisms of stress-related immunosuppression in the CNS.

Glutathione depletion associated with the HIV-1 TAT protein mediates the extracellular appearance of acidic fibroblast growth factor.

Primary murine embryonic fibroblasts transfected with HIV-1 TAT demonstrated decreased levels of high energy phosphates (ATP, GTP, UTP/CTP), adenine nucleotides (ATP, ADP, AMP), and both NAD+/NADH redox pairs, resulting in a substantial loss of redox poise. A greater than 50% decrease in intracellular reduced glutathione (GSH) concentration was accompanied by the extracellular appearance of acidic fibroblast growth factor (FGF-1). Addition of either N-acetyl-L-cysteine or glutathione ester (GSE), but not L-2-oxothiazolidine 4-carboxylate, partially restored intracellular GSH levels and resulted in loss of extracellular FGF-1. Treatment of FGF-1-transduced cells with buthionine sulfoximine (BSO) resulted in a time- and dose-dependent decrease in total cellular GSH concentration that was accompanied by the extracellular appearance of FGF-1. Inclusion of GSE during BSO treatment eliminated the extracellular appearance of FGF-1. BSO treatment of cells transfected with a mutant form of FGF-1, in which all three cysteine residues were replaced with serines, also decreased total cellular GSH concentration but failed to induce the extracellular appearance of FGF-1. Collectively, these results suggest that HIV-1 TAT induces a condition of oxidative stress, which mediates cellular secretion of FGF-1, an observation relevant to the pathophysiologic development and progression of AIDS-associated Kaposi's sarcoma.

Thiol redox modulation of tumor necrosis factor-alpha responsiveness in cultured AIDS-related Kaposi's sarcoma cells.

Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). While virtually every cell responds to TNF-alpha with gene activation, the extent of TNF-alpha-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-alpha membrane interaction, are part of this TNF-alpha-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-alpha. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-alpha challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV normal fibroblasts. In contrast, following TNF-alpha challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-alpha-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-alpha challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-alpha, suggest a biochemical rationale for the recognized TNF-alpha AIDS-KS clinical correlation.

Expression of interleukin-6 receptors and NF-kappa B in AIDS-related Kaposi sarcoma cell strains.

AIDS-related Kaposi sarcoma (AIDS-KS) is the most common malignancy associated with HIV infection, with an incidence of 10-30% of all AIDS patients. As such, there have been a large number of AIDS-KS cell strains isolated and numerous studies conducted to elucidate the mechanisms of malignancy in this disease. We have reported histological grade associated differences in the ability of AIDS-KS cell strains to proliferate under conditions of minimal growth factor supplementation, with strains derived from high grade lesions having enhanced proliferation potential. Furthermore, we found that this difference in in vitro growth characteristics was not attributed to grade associated differences in autologous growth factor release. These current investigations explored the hypothesis that grade associated growth differences could be attributed to differences in the expression of the components of the IL-6 receptor, or expression/inducibility of the pleotrophic transcription factor NF-kappa B. We determined there were no significant grade associated differences in the expression of either component (IL-6R alpha chain or gpl30) of the IL-6 receptor. However, non-lesional oral derived cell strain lysates from AIDS-KS patients (n = 4) contained significantly lower concentrations of both components of the IL-6 receptor than AIDS-KS strains (n = 8) and lower concentrations of gp-130 than normal human oral derived fibroblasts (n = 2). Comparative analysis of sera concentrations of soluble components of the IL-6 receptor did not demonstrate significant differences between HIV+/KS+ (n = 7), HIV+/KS- (n = 9) and normal (HIV-/KS-) (n = 4) populations. Further, no differences were detected in the expression of NF-kappa B in AIDS-KS cell strains (n = 5) derived from both high and low histological grade lesions as compared to nonlesional AIDS-KS cell stain (n = 1) and normal human oral derived fibroblasts (n = 2) under conditions of: constitutive/proliferative growth, sera starvation, oxidative stress, and mitogen reintroduction after sera starvation. In conclusion, these investigations have eliminated two explanations for histological grade associated differences for in vitro growth potential of AIDS-related KS cell strains and further substantiated the lack of systemic paracrine cytokine/cytokine receptor effects in AIDS-KS pathogenesis.

Mutated and wild-type p53 expression and HPV integration in proliferative verrucous leukoplakia and oral squamous cell carcinoma.

The frequencies of overexpression and mutation in the p53 tumor suppressor gene were examined in proliferative verrucous leukoplakia and oral squamous cell carcinoma with immunohistochemistry and single-strand conformation polymorphism analysis of DNA fragments amplified by polymerase chain reaction. Ten samples each of normal oral mucosa, proliferative verrucous leukoplakia, and squamous cell carcinoma were immunostained with antibodies against p53 protein; 8 of 10 cases of proliferative verrucous leukoplakia cases and 7 of 10 cases of oral squamous cell carcinoma were positive for p53 protein. Minimal staining was observed in normal oral tissues. The quantified labeling indexes demonstrated a range that corresponded to lesion progression. Single-strand conformation polymorphism analysis revealed p53 gene mutations within exons 5 to 8 in 40% (4 of 10) of the squamous cell carcinoma samples. Two of the 4 mutated squamous cell carcinoma samples lacked p53 expression. No p53 mutations were detected in proliferative verrucous leukoplakia tissues. Human papillomavirus 16 was identified in 2 of 7 p53 positive oral squamous cell carcinoma samples. Human papillomavirus 16 and 18 were identified in two of eight p53 positive proliferative verrucous leukoplakia samples. One p53 negative squamous cell carcinoma sample was positive for human papillomavirus 16 and had a mutation in exon 6 of the p53 gene. Human papillomavirus infection along with p53 expression plays a yet to be defined role in the pathogenesis of a limited number of cases of proliferative verrucous leukoplakia and squamous cell carcinoma. p53 immunohistochemistry, p53 gene mutations, and human papillomavirus infection prevalence do not provide a means to differentiate between leukoplakia and carcinoma and do not provide a predictive test for progression of leukoplakia to carcinoma.

Transforming growth factor-alpha overexpression in proliferative verrucous leukoplakia and oral squamous cell carcinoma: an immunohistochemical study.

Proliferative verrucous leukoplakia is a unique type of oral leukoplakia that has a high risk of malignant transformation. The aim of this study was to examine the expression of transforming growth factor-alpha in proliferative verrucous leukoplakia, oral squamous cell carcinoma, and normal mucosa. Transforming growth factor-alpha, a potent mitogen, is known to play an important role in various neoplasms including oral squamous cell carcinoma. Immunohistochemical localization of transforming growth factor-alpha in archival paraffin-embedded sections was performed with commercially available monoclonal antibodies. Ten cases each of normal mucosa, proliferative verrucous leukoplakia, and oral squamous cell carcinoma were stained. Quantification of the staining intensity, expressed as the cytoplasmic optical density, was done with the Roche Image Analysis System. The data were statistically analyzed with the one-way analysis of variance and Tukey tests. Notably, the mean cytoplasmic optical density of proliferative verrucous leukoplakia was significantly higher than the mean cytoplasmic optical density of normal mucosa (p < 0.01). The mean cytoplasmic optical density of proliferative verrucous leukoplakia was slightly higher than that of oral squamous cell carcinoma, however, this difference was not significant (p > 0.05). The mean cytoplasmic optical density values demonstrate that increased transforming growth factor-alpha immunoreactivity occurs in proliferative verrucous leukoplakia and oral squamous cell carcinoma relative to normal mucosa.

Cultured AIDS-related Kaposi's sarcoma (AIDS-KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: implication for oxidant stress in AIDS-KS pathogenesis.

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS-related Kaposi's sarcoma (AIDS-KS). However, it has been established that both cytokines, e.g. IL-6, and HIV-associated products, e.g., Tat, are integral in AIDS-KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor-kappa B[NF-kappa B] dependent routes) as well as the subsequent cytokine, tumor necrosis factor alpha (TNF alpha) stimulation of HIV replication. Features of AIDS-KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS-KS development. Time course nucleotide profile analyses show that AIDS-KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to 1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); 2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and 3) reduced levels of GSH (impeding both GSH peroxidase and GSH-S-transferases). Following exposure to physiologically relevant levels of H2O2, only the human microvascular endothelial cells (a putative AIDS-KS progenitor cell) responded with bioenergetic adaptations that reflected co-ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD+, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS-KS cells. Also, some of the AIDS-KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS-KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS-KS neoplastic development, or is it pre-existing in the population at risk? Regardless, use of antioxidant therapy (low risk/ potentially high benefit) in both the "at risk" population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality.

Comparison of constitutive cytokine release in high and low histologic grade AIDS-related Kaposi's sarcoma cell strains and in sera from HIV+/KS+ and HIV+/KS- patients.

Kaposi's sarcoma (KS) is both an AIDS-defining disease and the most common HIV-associated malignancy. A cytokine-mediated pathogenesis for AIDS-KS is implicated because AIDS-KS-derived cell strains both respond to and express a variety of cytokines. We have reported the establishment of several (n = 18) AIDS-KS cell strains and determined that reduced exogenous growth factors are necessary to sustain proliferation in isolates from high histologic grade KS lesions. This current investigation explored the possibility that there are histologic grade-associated differences in either the qualitative and/or quantitative constitutive release of AIDS-KS growth stimulatory cytokines. Our findings showed that the incorporation of HTLV-II cytokine-rich conditioned media induced both qualitative and significant quantitative cytokine release, suggesting that exogenous growth promoters stimulate constitutive cytokine release. ELISA of our AIDS-KS cell strains demonstrated constitutive release of IL-6 (seven of seven), FGF-2 (five of seven), GM-CSF (three of seven), and IL-1 beta (one of seven). None of our AIDS-KS cell strains constitutively released detectable levels of Onco-M, IL-4, PDGF, TNF-alpha, or TNF-beta. In addition, we report that the method of cytokine result quantitation significantly affects reported cytokine levels. We determined that there was no significant histologic grade-dependent difference in the constitutive release of soluble cytokines by in vitro grown cultures of AIDS-KS cells. The presence of HIV influenced the sera cytokine profiles by elevating IL-6 and decreasing PDGF concentrations of HIV+ individuals relative to HIV- healthy controls. However, the presence of KS was not associated with unique serum cytokine profiles, because no differences were noted in comparisons of HIV+/KS+ versus HIV+/KS- individuals. Our findings suggest that the local environment is key in modulating AIDS-KS cytokine expression and that KS growth-promoting factors function at the local or paracrine, not the systemic, level. In conclusion, our previous results demonstrated a histologic grade-associated difference in the in vitro growth capacity of AIDS-KS cells; with high histologic grade isolates displaying a marked growth advantage during culture in minimally supplemented media. Findings from this current study reveal that although the potential for a constitutive growth loop exists in the high-grade isolates, it is not reflected in the free levels of soluble cytokines secreted into the culture medium.

Human microvascular endothelial cell-extracellular matrix interaction in cellular growth state determination.

We introduce two methods, both of which are based on cellular-extracellular matrix interaction, which will facilitate the study of human microvascular endothelial cells. One method describes the means to obtain a G1 population baseline in human microvascular endothelial cells. Because of the contribution of the extracellular matrix in endothelial cell growth, synchronization in G1 was possible only after the incorporation of angiostatic levels of heparin and hydrocortisone into the extracellular matrix. In the second method, we demonstrate that selective perturbation of human microvascular endothelial cell-extracellular matrix interactions results in the induction of a transitional growth state, between proliferative and differentiated growth states, in human microvascular endothelial cells. In the functional, microtubule formation assays, transitional growth state endothelial cells display rates that are indermediate between those obtained from differentiated and proliferative endothelial cells. Our results demonstrate the importance of the human microvascular endothelial cell-extracellular matrix interaction in the determination of cellular growth state. Our findings also imply that responsiveness of microvascular endothelial cells to their cellular-extracellular matrix environs is highest during the differentiated growth state.

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities.

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained inflammatory states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levels of virus-induced cytokines and, because of a sparing effect on their phagocytic cells, retention of the phagocytic inflammatory response. A component of phagocytic activation is the initiation of the oxidative burst, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess decreased cytoprotective capabilities. Relative to either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplemented with conditioned medium from MOT, an HTLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed an inherent biochemical responsiveness, by increasing catalase activity following exposure to exogenous H2O2. In contrast, the catalase activity of AIDS-KS cells decreased following H2O2 challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression of AIDS-KS.

Environmental induction of tumor phenotype in a putative Kaposi sarcoma progenitor cell.

Many features of AIDS-related Kaposi sarcoma (AIDS-KS), e.g., multifocal lesional presentation at sites perfused by the microvasculature, suggest that AIDS-KS is initially a hyperplasia that subsequently progresses to a neoplasia. We propose that the unique AIDS environment, which contains high levels of circulating factors such as viral cytokines, is key in initiating the KS lesion. Further, we maintain that due to their physiological function, human microvascular endothelial cells (HMECs) are both likely target cells for the AIDS-related cytokines, and are putative AIDS-KS progenitor cells. Previously, we have shown that as a component of HMEC transition between proliferative and differentiated growth, HMECs modulate their nucleotide and glutathione levels. After attaining contact inhibition, HMECs enter a state of differentiation, which is characterized by cellular entrance into a G0, quiescent growth state, a decrease in cellular bioenergetic profiles, and spontaneous formation of microtubules. In contrast, when cultured in a "KS milieu", HMECs fail to differentiate. Instead, the "KS milieu" cultured cells assume a "growth relaxed" phenotype and demonstrate a lack of contact inhibition, loss of anchorage dependence, and retention of a "proliferative" bioenergetic profile despite culture confluence. Our results imply both that HMECs are responsive to AIDS-related cytokines, and that the local environment is key to instigating a relaxation of cellular growth controls.

Modulation of human microvascular endothelial cell bioenergetic status and glutathione levels during proliferative and differentiated growth.

During angiogenesis, formerly differentiated human microvascular endothelial cells (HMECs) return to a proliferative growth state. Many fundamental questions regarding HMEC function, such as how HMECs adapt to changes in bioenergetic requirements upon return to proliferative growth, remained unanswered. In this study, we evaluated whether modifications in HMEC bioenergetic profiles and glutathione (GSH) levels accompanied the cellular transition between differentiated and proliferative growth. To provide insight into the continuum of cellular adaptations that occur during this transition, we used a method recently developed in our laboratory that induces a state of morphological and functional predifferentiation in HMECs. Cellular morphology, in conjunction with flow cytometric DNA analyses and HMEC functional assays (the directed migration and intercellular association involved in microtubule formation) were employed to validate the HMEC culture state of growth. Analysis of the HPLC nucleotide profiles disclosed several findings common to all culture growth states. These uniform findings, e.g., cellular energy charges > 0.90, and highly reduced redox states, revealed that cultured HMECs maintain high rates of oxidative metabolism. However, there were also significant, culture growth state related differences in the nucleotide profiles. Proliferative HMECs were shown to possess significantly higher (relative to both large vessel endothelial cells, and differentiated HMECs) levels of GSH and specific nucleotides which were related with a return to the active cell cycle-ATP, GTP, UTP, and CTP, and NADPH. Further, the nucleotide profiles and GSH levels of the predifferentiated HMECs were determined to be intermediate between levels obtained for the proliferative and differentiated HMECs. The results of this study demonstrate that the capacity to modulate their cellular bioenergetic status during growth state transitions is one of the adaptations that enable HMECs to retain a growth state reciprocity. In addition, our findings also show that HMECs, especially during the proliferative growth state, are biochemically distinct from endothelial cells harvested from large vessels, and therefore suggest that HMECs are the cells of choice to employ when studying diseases that affect the human microvasculature.