Recombinant tissue plasminogen activator for the treatment of acute ischemic stroke.

The use of recombinant tissue plasminogen activator (rtPA) has been the standard of care for treatment of acute ischemic stroke for several years. Studies evaluating the efficacy, safety, and optimal timing of rtPA use are ongoing. Recently, results of new studies led to expansion of the short timeframe from stroke symptom onset in which a patient can receive this treatment. Additionally, more data are available to support the current goals of administration of rtPA to eligible patients as quickly as possible, as earlier treatment has been proven to improve outcomes.

Twist is required for thrombin-induced tumor angiogenesis and growth.

Twist, a master regulator of embryonic morphogenesis, induces functions that are also required for tumor invasion and metastasis. Because thrombin contributes to the malignant phenotype by up-regulating tumor metastasis, we examined its effect on Twist in five different tumor cell lines and two different endothelial cell lines. Thrombin up-regulated Twist mRNA and protein in all seven cell lines. Down-regulation of Twist in B16F10 tumor cell lines led to a approximately 3-fold decrease in tumor growth on a chorioallantoic membrane assay and approximately 2-fold decrease in syngeneic mice. Angiogenesis was decreased approximately 45% and 36%, respectively. The effect of Twist on angiogenesis was further examined and compared with the effect of thrombin. In studies using a Twist-inducible plasmid, several identical vascular growth factors and receptors were up-regulated approximately 2- to 3-fold in tumor cells as well as human umbilical vascular endothelial cells by both Twist as well as thrombin (vascular endothelial growth factor, KDR, Ang-2, matrix metalloproteinase 1, GRO-alpha, and CD31). Thrombin-induced endothelial cell chemotaxis and Matrigel endothelial cell tubule formation were similarly regulated by Twist. Thus, thrombin up-regulates Twist, which is required for thrombin-induced angiogenesis as measured by endothelial cell migration, Matrigel tubule formation, and tumor angiogenesis.

Thrombin up-regulates cathepsin D which enhances angiogenesis, growth, and metastasis.

Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin up-regulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3- to 8-fold and enhanced chemotaxis ( approximately 2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD. Secreted 4T1 CD induced neoangiogenesis by 2.4-fold on a chick chorioallantoic membrane, which was blocked in CD-KD cells. The addition of pure CD (2 ng) to the chick chorioallantoic membrane increased angiogenesis by 2.1-fold, which was completely inhibited by Pepstatin A (Pep A). CD enhanced human HUVEC chemotaxis and Matrigel tube formation by 2-fold, which was then blocked by Pep A. CD enhanced HUVEC matrix metalloproteinase 9 (MMP-9) activity by approximately 2-fold, which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001. The injection of CD-KD 4T1 cells into syngeneic mice inhibited tumor growth by 3- to 4-fold compared with empty vector (EV) cells. Hirudin, a specific thrombin inhibitor, inhibited the growth of wild-type and EV cells by 2- to 3-fold, compatible with thrombin up-regulation of CD. CD and thrombin also contributed to spontaneous pulmonary metastasis; 4-fold nodule inhibition with CD versus EV and 4.6-fold inhibition with hirudin versus EV (P < 0.02). Thus, thrombin-induced CD contributes to the malignant phenotype by inducing tumor cell migration, nodule growth, metastasis, and angiogenesis. CD-induced angiogenesis requires the proteolytic activation of MMP-9.

Evaluation of blood glucose values in critically ill patients before and after implementation of an intensive insulin infusion protocol.

This retrospective study evaluated the effect of an intensive insulin infusion protocol on blood glucose values in five intensive care units at Baylor University Medical Center. The protocol involved an equation in which the hourly blood glucose value and an adjusted multiplier were used to determine the insulin infusion rate. The default target blood glucose range was 90 to 120 mg/dL. Results showed that blood glucose values taken by diabetic fingerstick were significantly better in March 2006, after initiation of the protocol, than in March 2005, before use of the protocol, for the percentage of patients both with a blood glucose value >150 mg/dL (P < 0.001) and with a blood glucose value >120 mg/dL (P < 0.001). The percentage of patients with a blood glucose value ≤80 mg/dL was not significantly different between the two time periods (P > 0.10). The increased number of diabetic fingerstick values within a desired range was achieved without a significantly higher number of blood glucose values ≤80 mg/dL. It can be theorized that wide use of the protocol was at least partly responsible for the significant change in blood glucose values.

Disruption of endothelial cell interactions with the novel HU177 cryptic collagen epitope inhibits angiogenesis.

PURPOSE: The importance of cellular communication with the extracellular matrix in regulating cellular invasion is well established. Selective disruption of communication links between cells and the local microenvironment by specifically targeting non-cellular matrix-immobilized cryptic extracellular matrix epitopes may represent an effective new clinical approach to limit tumor-associated angiogenesis. Therefore, we sought to determine whether the HU177 cryptic collagen epitope plays a functional role in regulating angiogenesis in vivo. EXPERIMENTAL DESIGN: We examined the expression and characterized the HU177 cryptic collagen epitope in vitro and in vivo using immunohistochemistry and ELISA. We examined potential mechanisms by which this cryptic collagen epitope may regulate angiogenesis using in vitro cell adhesion, migration, proliferation, and biochemical assays. Finally, we examined the whether blocking cellular interactions with the HU177 cryptic epitope plays a role in angiogenesis and tumor growth in vivo using the chick embryo model. RESULTS: The HU177 cryptic epitope was selectively exposed within tumor blood vessel extracellular matrix, whereas little was associated with quiescent vessels. An antibody directed to this cryptic site selectively inhibited endothelial cell adhesion, migration, and proliferation on denatured collagen type IV and induced increased levels of cyclin-dependent kinase inhibitor p27(KIP1). Systemic administration of mAb HU177 inhibited cytokine- and tumor-induced angiogenesis in vivo. CONCLUSIONS: We provide evidence for a new functional cryptic regulatory element within collagen IV that regulates tumor angiogenesis. These findings suggest a novel and highly selective approach for regulating angiogenesis by targeting a non-cellular cryptic collagen epitope.

Inhibition of angiogenesis and tumor metastasis by targeting a matrix immobilized cryptic extracellular matrix epitope in laminin.

Angiogenesis and tumor metastasis depend on extracellular matrix (ECM) remodeling and subsequent cellular interactions with these modified proteins. An in-depth understanding of how both endothelial and tumor cells use matrix-immobilized cryptic ECM epitopes to regulate invasive cell behavior may lead to the development of novel strategies for the treatment of human tumors. However, little is known concerning the existence and the functional significance of cryptic laminin epitopes in regulating angiogenesis and tumor cell metastasis. Here, we report the isolation and characterization of a synthetic peptide that binds to a cryptic epitope in laminin. The STQ peptide selectively bound denatured and proteolyzed laminin but showed little interaction with native laminin. The cryptic laminin epitope recognized by this peptide was selectively exposed within malignant melanoma in vivo, whereas little if any was detected in normal mouse skin. Moreover, the STQ peptide selectively inhibited endothelial and tumor cell adhesion, migration, and proliferation in vitro and inhibited angiogenesis, tumor growth, and experimental metastasis in vivo. This inhibitory activity was associated with a selective up-regulation of the cyclin-dependent kinase inhibitor P27(KIP1) and induction of cellular senescence. These novel findings suggest the existence of functionally relevant cryptic laminin epitopes in vivo and that selective targeting of these laminin epitopes may represent an effective new strategy for the treatment of malignant tumors by affecting both the endothelial and tumor cell compartments.

The cochaperone p23 differentially regulates estrogen receptor target genes and promotes tumor cell adhesion and invasion.

The cochaperone p23 plays an important role in estrogen receptor alpha (ER) signal transduction. In this study, we investigated how p23 regulates ER target gene activation and affects tumor growth and progression. Remarkably, we found that changes in the expression of p23 differentially affected the activation of ER target genes in a manner dependent upon the type of DNA regulatory element. p23 overexpression enhanced the expression of the ER target genes cathepsin D and pS2, which are regulated by direct DNA binding of ER to estrogen response elements (ERE). In contrast, the expression of other target genes, including c-Myc, cyclin D1, and E2F1, to which ER is recruited indirectly through its interaction with other transcription factors remains unaffected by changes in p23 levels. The p23-induced expression of pS2 is associated with enhanced recruitment of ER to the ERE in the promoter, whereas ER recruitment to the ERE-less c-Myc promoter does not respond to p23. Intriguingly, p23-overexpressing MCF-7 cells exhibit increased adhesion and invasion in the presence of fibronectin. Our findings demonstrate that p23 differentially regulates ER target genes and is involved in the control of distinct cellular processes in breast tumor development, thus revealing novel functions of this cochaperone.

Inhibition of experimental metastasis by targeting the HUIV26 cryptic epitope in collagen.

Metastasis from the primary tumor to distant sites involves an array of molecules that function in an integrated manner. Proteolytic remodeling and subsequent tumor cell interactions with the extracellular matrix regulate tumor invasion. In previous studies, we have identified a cryptic epitope (HUIV26) that is specifically exposed after alterations in the triple helical structure of type IV collagen. Exposure of this cryptic epitope plays a fundamental role in the regulation of angiogenesis in vivo. However, little is known concerning the ability of tumor cells to interact with this cryptic site or whether this site regulates tumor cell metastasis in vivo. In this regard, many of the same cellular processes that regulate angiogenesis also contribute to tumor metastasis. Here we provide evidence that tumor cells such as B16F10 melanoma interact with denatured collagen type IV in part by recognizing the HUIV26 cryptic site. Systemic administration of a HUIV26 monoclonal antibody inhibited experimental metastasis of B16F10 melanoma in vivo. Taken together, our findings suggest that tumor cell interactions with the HUIV26 cryptic epitope play an important role in regulating experimental metastasis and that this cryptic element may represent a therapeutic target for controlling the spread of tumor cells to distant sites.

Recombinant alpha2(IV)NC1 domain inhibits tumor cell-extracellular matrix interactions, induces cellular senescence, and inhibits tumor growth in vivo.

Cellular interaction with the extracellular matrix is thought to be a critical event in controlling angiogenesis and tumor growth. In our previous studies, genetically distinct noncollagenous (NC) domains of type-IV collagen were shown to interact with integrin receptors expressed on the surface of endothelial cells. Moreover, these NC1 domains were shown to inhibit angiogenesis in vivo. Here, we provide evidence that a recombinant form of the alpha2(IV)NC1 domain of type-IV collagen could bind integrins alpha1beta1 and alphavbeta3 expressed on melanoma cells and inhibit tumor cell adhesion in a ligand-specific manner. Systemic administration of recombinant alpha2(IV)NC1 domain potently inhibited M21 melanoma tumor growth within full thickness human skin and exhibited a dose-dependent inhibition of tumor growth in nude mice. Interestingly, alpha2(IV)NC1 domain enhanced cellular senescence in tumor cells in vitro and in vivo. Taken together, these results suggest that recombinant alpha2(IV)NC1 domain is not only a potent anti-angiogenic reagent, but it also directly impacts tumor cell behavior. Thus, alpha2(IV)NC1 domain represents a potent inhibitor of tumor growth by impacting both endothelial and tumor cell compartments.

Ionizing radiation modulates the exposure of the HUIV26 cryptic epitope within collagen type IV during angiogenesis.

PURPOSE: The majority of the research on the biologic effects of ionizing radiation has focused on the impact of radiation on cells in terms of gene expression, DNA damage, and cytotoxicity. In comparison, little information is available concerning the direct effects of radiation on the extracellular microenvironment, specifically the extracellular matrix and its main component, collagen. We have developed a series of monoclonal antibodies that bind to cryptic epitopes of collagen Type IV that are differentially exposed during matrix remodeling and are key mediators of angiogenesis. We have hypothesized that ionizing radiation might affect the process of angiogenesis through a direct effect on the extracellular matrix and specifically on collagen Type IV. METHODS AND MATERIALS: Angiogenesis was induced in a chick chorioallantoic membrane (CAM) model; 24 h later, a single-dose treatment with ionizing radiation (0.5, 5, and 20 cGy) was administered. Angiogenesis was assessed, and the exposure of two cryptic regulatory epitopes within collagen Type IV (HUI77 and HUIV26) was studied in vitro by solid-phase ELISA and in vivo by immunofluorescence staining. RESULTS: A dose-dependent reduction of angiogenesis with maximum inhibition (85%-90%) occurring at 20 cGy was demonstrated in the CAM model. Exposure of the cryptic HUIV26 site, an angiogenesis control element, was inhibited both in vitro and in vivo by the same radiation dose, whereas little if any change was observed for the HUI77 cryptic epitope. CONCLUSIONS: A dose-dependent alteration of the functional exposure of the HUIV26 cryptic epitope is induced by radiation in vitro and in the CAM model in vivo. This radiation-induced change in protein structure and function may contribute to the inhibitory effects of ionizing radiation on new blood vessel growth and warrants further studies in other models.