FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells.

We previously found that FoxM1B is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly angiogenic glioblastoma in nude mice. However, the molecular mechanisms by which FoxM1B enhances glioma angiogenesis are currently unknown. In this study, we found that vascular endothelial growth factor (VEGF) is a direct transcriptional target of FoxM1B. FoxM1B overexpression increased VEGF expression, whereas blockade of FoxM1 expression suppressed VEGF expression in glioma cells. Transfection of FoxM1 into glioma cells directly activated the VEGF promoter, and inhibition of FoxM1 expression by FoxM1 siRNA suppressed VEGF promoter activation. We identified two FoxM1-binding sites in the VEGF promoter that specifically bound to the FoxM1 protein. Mutation of these FoxM1-binding sites significantly attenuated VEGF promoter activity. Furthermore, FoxM1 overexpression increased and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma cells. Finally, an immunohistochemical analysis of 59 human glioblastoma specimens also showed a significant correlation between FoxM1 overexpression and elevated VEGF expression. Our findings provide both clinical and mechanistic evidence that FoxM1 contributes to glioma progression by enhancing VEGF gene transcription and thus tumor angiogenesis.

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

NF-kappaB and AP-1 connection: mechanism of NF-kappaB-dependent regulation of AP-1 activity.

Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Molecular mechanisms of response in gastrointestinal stromal tumors treated with imatinib mesylate

Gastrointestinal stromal tumors (GIST) represent 80% of sarcoma arising from the GI tract. The inciting event in tumor progression is mutation of the kit or, rarely, platelet derived growth factor receptor-α (PDGFR) gene. These mutations encode ligand independent, constitutively active proteins: Kit or PDGFR. ^ These tumors are notoriously chemo and radio resistant. Historically, patients with advanced disease realized a median overall survival of 9 months. However, with modern management of GIST with imatinib mesylate (Novartis), a small molecule inhibitor of the Kit, PDGFR, and Abl tyrosine kinases, patients now realize a median overall survival greater than 30 months. However, almost half of patients present with surgically resectable GIST and the utility of imatinib in this context has not been prospectively studied. Also, therapeutic benefit of imatinib is variable from patient to patient and alternative targeted therapy is emerging as potential alternatives to imatinib. Thus, elucidating prognostic factors for patients with GIST in the imatinib-era is crucial to providing optimal care to each particular patient. Moreover, the exact mechanism of action of imatinib in GIST is not fully understood. Therefore, physicians find difficulty in accurately predicting which patient will benefit from imatinib, how to assess response to therapy, and the time at which to assess response. ^ I have hypothesized that imatinib is tolerable and clinically beneficial in the context of surgery, VEGF expression and kit non-exon 11 genotypes portend poor survival on imatinib therapy, and imatinib's mechanism of action is in part due to anti-vascular effects and inhibition of the Kit/SCF signaling axis of tumor-associated endothelial cells. ^ Results herein demonstrate that imatinib is safe and increases the duration of disease-free survival when combined with surgery. Radiographic and molecular (namely, apoptosis) changes occur within 3 days of imatinib initiation. I illustrate that non-exon 11 mutant genotypes and VEGF are poor prognostic factors for patients treated with imatinib. These findings may allow for patient stratification to emerging therapies rather than imatinib. I show that imatinib has anti-vascular effects via inducing tumor endothelial cell apoptosis perhaps by abrogation of the Kit/SCF signaling axis. ^

The effects of proteasome inhibition on angiogenesis and autophagy in human prostate cancer cells

The proteasome degrades approximately 80% of intracellular proteins to maintain homeostasis. Proteasome inhibition is a validated therapeutic strategy, and currently, proteasome inhibitor bortezomib is FDA approved for the treatment of MM and MCL. Specific pathways affected by proteasome inhibition have been identified, but mechanisms of the anti-tumor effects of proteasome inhibition are not fully characterized and cancer cells display marked heterogeneity in terms of their sensitivity to proteasome inhibitor induced cell death. ^ The antitumor effects of proteasome inhibition involve suppression of tumor angiogenesis and vascular endothelial growth factor (VEGF) expression, but the mechanisms involved have not been clarified. In this dissertation I investigated the mechanisms underlying the effects of two proteasome inhibitors, bortezomib and NPI-0052, on VEGF expression in human prostate cancer cells. I found that proteasome inhibitors selectively downregulated hypoxia inducible factor 1alpha (HIF-1α) protein and its transcriptional activity to inhibit VEGF expression. Mechanistic studies demonstrated that proteasome inhibitors mediate the induction of the unfolded protein response (UPR) and that downregulation of HIF-1α is caused by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and translation repression. Importantly, I showed that proteasome inhibitors activated the UPR in some cells but not in others. My observation may have implications for the design of combination regimens that are based on exploiting proteasome inhibitor-induced ER stress.^ Although proteasome inhibitors have shown modest activity on prostate cancer, there is general consensus that no single agent is likely to have significant activity in prostate cancer. In the second part of this dissertation I attempted to exploit the effects of proteasome inhibition on the UPR to design a combination therapy that would enhance cancer cell death. Autophagy is a lysosome dependent degradation pathway that functions to eliminate long-lived protein and subcellular structures. Targeting autophagy has been shown to inhibit tumors in preclinical studies. I found that inhibition of autophagy with chloroquine or 3-methyladenine enhanced proteasome inhibitor induced cell death and the effects were associated with increased intracellular stress as marked by aggresome formation. Multiple cancers appear to be resistant to proteasome inhibition treatment alone. The implications of synergy for the combined inhibition of autophagy and the proteasome would likely apply to other cancers aside from prostate cancer. ^

Role of IkappaB kinase beta in inflammation-mediated breast cancer development

Breast cancer is the second most common farm of cancers and the second leading cause of cancer death for American women. Clinical studies indicate inflammation is a risk factor for breast cancer development. Among the cytokines and chemokines secreted by the infiltrating inflammatory cells, tumor necrosis factor a (TNFα) is considered one of the most important inflammatory factors involved in inflammation-mediated tumorigenesis. ^ Here we found that TNFα/IKKβ signaling pathway is able to increase tumor angiogenesis through activation of mTOR pathway. While investigating which molecule in the mTOR pathway involved in TNFα/IKKβ-mediated mTOR activation, our results showed that IKKβ physically interacts with and phosphorylates TSC1 at Ser487 and Ser511 in vitro and in vivo. Phosphorylation of TSC1 by IKKβ inhibits its association with TSC2, alters TSC2 membrane localization, and thereby activates mTOR. In vitro angiogenesis assays and orthotopic breast cancer model reveals that phosphorylation of TSC1 by IKKβ enhances VEGF expression, angiogenesis and culminates in tumorigenesis. Furthermore, expression of activated IKKβ is associated with TSC1 Ser511 phosphorylation and VEGF production in multiple tumor types and correlates with poor clinical outcome of breast cancer patients. ^ Furthermore, dysregulation of tumor suppressor FOXO3a contributes to the development of breast cancer. We found that overexpression of IKKβ led to inhibition of FOXO3a-mediated transactivation activity. While investigating the underlying mechanisms of IKKβ-mediated dysregulation of FOXO3a, our results showed that IKKβ physically associated with FOXO3a and phosphorylated FOXO3a at Ser644 in vitro and in vivo. The phosphorylation of FOXO3a by IKKβ altered its subcellular localization from nucleus to cytoplasm and promoted its degradation through ubiquitin-proteasome pathway. Mutation of FOXO3a at Ser644 prevented IKKβ-induced ubiquitination and degradation. In vitro cell proliferation assay and orthotopic breast cancer model revealed that phosphorylation of FOXO3a by IKKβ overrode FOXO3a-mediated repression of tumor progression. ^ In conclusion, our findings identify IKKβ-mediated suppressions of both TSC1 and FOXO3a are critical for inflammation-mediated breast cancer development through increasing tumor angiogenesis and evading apoptosis, respectively. Understanding the role of IKKβ in both FOXO3a and TSC/mTOR signaling pathways provides a critical insight of inflammation-mediated diseases and may provide a target for clinical intervention in human breast cancer. ^

The critical role and regulation of transcription factor FoxM1 in gastric cancer development and progression

The mammalian Forkhead Box (Fox) transcription factor (FoxM1) is implicated in tumorgenesis. However, the role and regulation of FoxM1 in gastric cancer remain unknown.^ I examined FoxM1 expression in 86 cases of primary gastric cancer and 57 normal gastric tissue specimens. I found weak expression of FoxM1 protein in normal gastric mucosa, whereas I observed strong staining for FoxM1 in tumor-cell nuclei in various gastric tumors and lymph node metastases. The aberrant FoxM1 expression is associated with VEGF expression and increased angiogenesis in human gastric cancer. A Cox proportional hazards model revealed that FoxM1 expression was an independent prognostic factor in multivariate analysis. Furthermore, overexpression of FoxM1 by gene transfer significantly promoted the growth and metastasis of gastric cancer cells in orthotopic mouse models, whereas knockdown of FoxM1 expression by small interfering RNA did the opposite. Next, I observed that alteration of tumor growth and metastasis by elevated FoxM1 expression was directly correlated with alteration of VEGF expression and angiogenesis. In addition, promotion of gastric tumorigenesis by FoxM1 directly and significantly correlated with transactivation of vascular endothelial growth factor (VEGF) expression and elevation of angiogenesis. ^ To further investigate the underlying mechanisms that result in FoxM1 overexpression in gastric cancer, I investigated FoxM1 and Krüppel-like factor 4 (KLF4) expressions in primary gastric cancer and normal gastric tissue specimens. Concomitance of increased expression of FoxM1 protein and decreased expression of KLF4 protein was evident in human gastric cancer. Enforced KLF4 expression suppressed FoxM1 protein expression. Moreover, a region within the proximal FoxM1 promoter was identified to have KLF4-binding sites. Finally, I found an increased FoxM1 expression in gastric mucosa of villin-Cre -directed tissue specific Klf4-null mice.^ In summary, I offered both clinical and mechanistic evidence that dysregulated expression of FoxM1 play an important role in gastric cancer development and progression, while KLF4 mediates negative regulation of FoxM1 expression and its loss significantly contributes to FoxM1 dysregulation. ^

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Conditional expression of Sry, and Wnt4 by gene-targeting: Studies in sexual and skeletal development

Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) locus by gene targeting in embryonic stem (ES) cells to create a conditional gene expression system in mice. In the targeted alleles, expression of these cDNAs should be blocked by a neomycin resistance selection cassette that is flanked by loxP sites. Transgene expression should be activated after the blocking cassette is deleted by Cre recombinase. ^ To test this conditional expression system, I have bred R26-stop- Sry and R26-stop-Wnt4 heterozygotes with a MisRII-Cre mouse line that expresses Cre in the gonads of both sexes. Analysis of these two types of bigenic heterozygotes indicated that their gonads developed normally like those of wild types. However, one XX R26-Sry/R26-Sry; MisR2-Cre/+ showed epididymis-like structures resembling those of males. In contrast, only normal phenotypes were observed in XY R26-Wnt4/R26-Wnt4; MisR2-Cre /+ mice. To interpret these results, I have tested for Cre recombinase activity by Southern blot and transcription of the Sry and Wnt4 transgenes by RT-PCR. Results showed that bigenic mutants had insufficient activation of the transgenes in their gonads at E12.5 and E13.5. Therefore, the failure to observe mutant phenotypes may have resulted from low activity of MisR2-Cre recombination at the appropriate time. ^ Col2a1-Cre transgenic mice express Cre in differentiating chondrocytes. R26-Wnt4; Col2a1-Cre bigenic heterozygous mice were found to exhibit a dramatic alteration in growth presumably caused by Wnt4 overexpression during chondrogenesis. R26-Wnt4; Col2a1-Cre mice exhibited dwarfism beginning approximately 10 days after birth. In addition, they also had craniofacial abnormalities, and had delayed ossification of the lumbar vertebrate and pelvic bones. Histological analysis of the growth plates of R26-Wnt4; Col2a1-Cre mice revealed less structural organization and a delay in onset of the primary and secondary ossification centers. Molecular studies confirmed that overexpression of Wnt4 causes decreased proliferation and early maturation of chondrocytes. In addition, R26-Wnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF), suggesting that defects in vascularization may contribute to the dwarf phenotype. Finally, 9-month-old R26-Wnt4; Col2a1-Cre mice had significantly more fat cells in the marrow cavities of their metaphysis long bones, implying that long-term overexpression of Wnt4may cause bone marrow pathologies. In conclusion, Wnt4 was activated by Col2a1-Cre recombinase and was overexpressed in the growth plate, resulting in aberrant proliferation and differentiation of chondrocytes, and ultimately leads to dwarfism in mice. ^