A novel Bruton's tyrosine kinase inhibitor CC-292 in combination with the proteasome inhibitor carfilzomib impacts the bone microenvironment in a multiple myeloma model with resultant antimyeloma activity.

Bruton's tyrosine kinase (Btk) modulates B-cell development and activation and has an important role in antibody production. Interestingly, Btk may also affect human osteoclast (OC) function; however, the mechanism was unknown. Here we studied a potent and specific Btk inhibitor, CC-292, in multiple myeloma (MM). In this report, we demonstrate that, although CC-292 increased OC differentiation, it inhibited OC function via inhibition of c-Src, Pyk2 and cortactin, all involved in OC-sealing zone formation. As CC-292 did not show potent in vitro anti-MM activity, we next evaluated it in combination with the proteasome inhibitor, carfilzomib. We first studied the effect of carfilzomib on OC. Carfilzomib did not have an impact on OC-sealing zone formation but significantly inhibited OC differentiation. CC-292 combined with carfilzomib inhibited both sealing zone formation and OC differentiation, resulting in more profound inhibition of OC function than carfilzomib alone. Moreover, the combination treatment in an in vivo MM mouse model inhibited tumor burden compared with CC-292 alone; it also increased bone volume compared with carfilzomib alone. These results suggest that CC-292 combined with carfilzomib augments the inhibitory effects against OC within the bone microenvironment and has promising therapeutic potential for the treatment of MM and related bone disease.

An inhibitor of methionine aminopeptidase type-2, PPI-2458, ameliorates the pathophysiological disease processes of rheumatoid arthritis.

OBJECTIVE: To elucidate the role of methionine aminopeptidase type-2 (MetAP-2) in the clinical pathology of rheumatoid arthritis, arthritis was induced in rats by administration of peptidoglycan-polysaccharide (PG-PS). DESIGN: The inhibitor of MetAP-2, PPI-2458, was administered orally at 5 mg/kg every other day during 3 distinct phases of the disease. In vitro studies were performed to clarify in vivo findings. RESULTS: Ankle swelling was completely alleviated by MetAP-2 inhibition. Inhibition of MetAP-2 in blood and tissues correlated with protection against PG-PS-induced arthritis. Histopathology of the tarsal joints improved following PPI-2458 administration, including a significant improvement of bone structure. In in vitro studies, osteoclast formation and activity were inhibited by PPI-2458, a mechanism not previously attributed to MetAP-2 inhibition. CONCLUSIONS: The important role that MetAP-2 has in the pathophysiological disease processes of PG-PS arthritis provides a strong rationale for evaluating PPI-2458 as a disease modifying antirheumatic treatment for rheumatoid arthritis.

Integrins as targets of angiogenesis inhibition.

Integrins area widely distributed family of cell surface alpha/beta heterodimers that bind cells to components of the extracellular matrix and mediate cell-cell interactions. Integrin alpha(v)beta3 interacts with RGD (Arg-Gly-Asp) sequence-containing proteins in the extracellular matrix. The distribution of alpha(v)beta3 is highly restricted, with expression on activated endothelium, activated vascular smooth muscle, tumors, and osteoclasts. Expression of alpha(v)beta3 may contribute to a malignant phenotype by supporting the growth and persistence of small blood vessels that nourish the primary and metastatic tumors and increasing invasive potential. Inhibition of alpha(v)beta3 can modulate tumor-induced angiogenesis and can increase apoptosis of tumor-associated small blood vessels. It might also help control humoral hypercalcemia of malignancy through direct or indirect activity on the osteoclast. Preclinical studies found that several RGD peptidomimetics and a monoclonal antibody to alpha(v)beta3 can inhibit tumor growth by blocking tumor-induced angiogenesis.

Magnetic resonance contrast enhancement of neovasculature with alpha(v)beta(3)-targeted nanoparticles.

Site-directed contrast enhancement of angiogenic vessels in vivo was demonstrated using antibody targeting of an MRI contrast agent to the alpha(v)beta(3) integrin, a molecular marker characteristic of angiogenic endothelium. The agent was tested in a rabbit corneal micropocket model, in which neovasculature is induced in the cornea using basic fibroblast growth factor. The targeted contrast agent consists of Gd-perfluorocarbon nanoparticles linked to alpha(v)beta(3) integrin antibody DM101. The animal group receiving the targeted contrast agent displayed a 25% increase in the average MR signal intensity after 90 min. Control groups in which the nanoparticles are either used alone, linked to an isotype-matched antibody, or linked to DM101 and administered following receptor blocking did not display MR contrast enhancement at similar dose levels. These findings indicate that the antibody-targeted agent enhances MR signal intensity in the capillary bed in a corneal micropocket model of angiogenesis, and is selectively retained within the angiogenic region via specific interaction with the alpha(v)beta(3) epitope.

Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

Characterization of spontaneous metastasis in an aggressive breast carcinoma model using flow cytometry.

Studies of metastasis can be accelerated and provide more mechanistic information using cell lines which reproducibly and aggressively metastasize, and which are accurately and easily detected in tissues at all stages of the metastatic process. Although reporter proteins such as green fluorescent protein (GFP) and beta-galactosidase have improved the tracking of tumor cells in vivo, their measurement has often been limited to visual observation and manual counting. In this study, we exploited the highly sensitive and objective quantitation provided by flow cytometry to characterize, in detail, the sequence of events associated with orthotopic metastasis in a highly aggressive mouse model. Following stable transfection of the MDA-MB-435 breast carcinoma cell line with GFP, we utilized an in vivo selection process to isolate a variant exhibiting increased primary tumor growth and metastasis. As few as one fluorescent tumor cell per 200,000 host cells could be accurately detected in dissociated tissues by flow cytometry, allowing us to demonstrate that metastatic cells migrate to the lungs of SCID mice very early after orthotopic implantation. Tumor burden in lungs increased in a smooth continuous manner, until death approximately eight weeks later. Levels of circulating tumor cells in blood were also detectable at an early timepoint, but remained relatively low throughout the course of secondary tumor development in the lungs. Surgical removal of the primary tumor at various times after inoculation significantly affected lung tumor burden, supporting the concept that circulating tumor cells in blood inefficiently initiate distal metastases. Furthermore, the continuing contribution to metastasis by the primary tumor was independent of tumor mass. The combined characteristics of enhanced orthotopic metastasis and quantitative detection in blood and tissues will make this a useful new model for the characterization of the multi-stage progression of cancer, and the preclinical evaluation of anti-neoplastic therapies.

A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy.

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.

Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.

We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability to associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites. A detergent based extraction and Western blotting procedure demonstrated an association of E-selectin with the insoluble (cytoskeletal) fraction of endothelial monolayers that correlated with adhesion of leukocytes via an E-selectin-dependent mechanism. A mutant form of E-selectin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cell and supported leukocyte attachment (in a nonstatic adhesion assay) in a fashion similar to the native E-selectin molecule, but failed to become associated with the cytoskeletal fraction. To identify the cytoskeletal components that associate with the cytoplasmic domain of E-selectin, paramagnetic beads coated with the adhesion-blocking anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alpha-actinin, vinculin, filamin, paxillin, as well as focal adhesion kinase (FAK), were copurified by this procedure, however talin was not. When a mechanical stress was applied to H18/7-coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoskeletal mechanical linkage. COS-7 cells transfected with the tailless E-selectin failed to show resistance to the twisting stress. Taken together, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin through its cytoplasmic domain, a process which may have important implications for cell-cell signaling as well as mechanical anchoring during leukocyte-endothelial adhesive interactions.

Neutrophil-mediated damage to human vascular endothelium. Role of cytokine activation.

Cytokine activation of cultured human vascular endothelial cells renders them hyperadhesive for blood leukocytes. Co-incubation of freshly isolated, unstimulated human blood neutrophils with confluent cytokine-activated human endothelial monolayers for 90 minutes results in extensive endothelial detachment and destruction of monolayer integrity. In contrast, unactivated endothelial monolayers remain intact. Using this in vitro model, we have explored the neutrophil-effector mechanisms involved in this injury. Coincubation in the presence of a serine protease inhibitor (phenylmethylsulfonyl fluoride) or specific elastase inhibitors (Ala-Ala-Pro-Val-chloromethyl ketone or alpha-1-protease inhibitor) markedly diminished injury. In contrast, scavengers or inhibitors of oxygen-derived free radicals (superoxide dismutase, catalase, mannitol, or sodium azide) were not protective. Purified human neutrophil elastase mimicked the effect of the neutrophils suggesting a key role for elastase in the neutrophil-mediated injury in this model. Interfering with direct neutrophil-endothelial cell contact by interposing a microporous barrier insert prevented endothelial cell detachment. Furthermore, this neutrophil-mediated detachment could be inhibited with interleukin-8, an action correlated with a decrease in neutrophil adhesion to activated endothelial monolayers. By defining the role of endothelial activation in neutrophil-mediated injury, this in vitro model may provide useful insights into potential therapeutic interventions designed to prevent disruption of the endothelial barrier function.

Interleukin-8 induces changes in human neutrophil actin conformation and distribution: relationship to inhibition of adhesion to cytokine-activated endothelium.

Interleukin-8 (IL-8) induces diverse biological responses in neutrophils, including inhibition of adhesion to cytokine-activated endothelium, which we have termed the leukocyte adhesion inhibition (LAI) effect. Pretreatment of neutrophils with cytochalasin B abolished the LAI effect of IL-8, suggesting a microfilament-dependent mechanism. Interleukin-8 induced a rapid increase (less than or equal to 15 s) in the polymerization of actin filaments in human neutrophils that was blocked by pretreatment with cytochalasin B. F-actin depolymerization occurred gradually at a rate inversely proportional to IL-8 concentration. This temporal pattern of actin polymerization-depolymerization was similar to that induced by other chemotactic factors such as C5a and N-formylmethionyl-leucyl-phenylalanine, which also exhibit a marked LAI effect, but the lipid mediators, leukotriene B4 and platelet-activating factor, lack any significant LAI effect. Scanning confocal microscopy demonstrated that neutrophil actin microfilaments undergo a dramatic rearrangement prior to detachment of the neutrophil from a surface. We suggest that the ability of IL-8 and certain other leukocyte agonists to regulate the actin polymer network of neutrophils may play an important role in adhesive interactions with the vascular endothelium.

Exaggerated renal thromboxane and prostaglandin release by angiotensin II in suprarenal aortic coarctation hypertension.

The ability of angiotensin II and arachidonic acid to release immunoreactive prostaglandins into venous and ureteral effluents of rabbit isolated perfused kidneys was examined 7 days after suprarenal aortic coarctation (SRAC) or sham operation (SHAM). Renal vascular responses to angiotensin II were significantly enhanced in SRAC and accompanied by an enhanced venous efflux of bioassayable prostaglandins. Angiotension II-induced release of immunoreactive PGE2, PGF2 alpha, 6-keto PGF1 alpha and TxB2 into the venous effluent was exaggerated in SRAC. As angiotensin II did not stimulate TxB2 efflux in the SHAM group the induction of TxB2 release by SRAC is particularly noteworthy. These changes in eicosanoid release in response to angiotensin II were not mimicked by arachidonic acid administration. These results suggest that in renovascular hypertension angiotensin II-induced prostaglandin release is primarily augmented in the vascular compartment and is consistent with the sensitivity of renal function to cyclooxygenase inhibitors in renovascular hypertension.

Glandular kallikrein levels in the rat anterior pituitary during the estrous cycle and pregnancy.

Glandular kallikrein (a trypsin-like serine protease) is a major estrogen-induced protein in the rat anterior pituitary, which appears to be associated with lactotropes. The present study examined glandular kallikrein levels in the anterior pituitary during the rat estrous cycle and pregnancy. After trypsin treatment of anterior pituitary homogenates (to activate latent forms of the enzyme), glandular kallikrein activity was measured by using the chromogenic substrate D-val-leu-arg-p-nitroanilide: 98-95% of the enzymatic activity was immunoprecipitable with glandular kallikrein antiserum. Glandular kallikrein levels did not change significantly during the various phases of the rat estrous cycle. However, a sharp decrease was observed starting on Day 15 of pregnancy and lasting through parturition; levels had almost returned to control values by Day 5 of lactation.

Renal prostaglandin efflux induced by vasopressin, dDAVP and arachidonic acid: contrasting profile and sites of release.

Prostaglandin (PG) efflux into ureteral (UE) and venous effluents (VE) of rabbit isolated perfused kidneys was determined by superfusion bioassay and radioimmunoassay (RIA), in response to injections of arginine-vasopressin (AVP), the non-pressor vasopressin analogue 1-deamino-8-D-arginine vasopressin (dDAVP) and arachidonic acid (AA). dDAVP (10-1000 ng) failed to stimulate renal PG release, whereas AVP (10-100 ng) and AA (10-50 micrograms) caused a dose-dependent release of PG. AVP evoked PG release into both effluents with release into the VE greater than UE at high doses. In contrast, PG release by AA was almost exclusively into the VE. Indomethacin (2.8 X 10(-6) mol/l) abolished AVP- and AA-induced PG efflux in both effluents, and vasodepressor responses to AA. PGE2 was the predominant PG released in response to AVP in both effluents whereas AA released primarily 6-keto-PGF1 alpha. The contrasting sites and profile of released PG suggest that exogenous AA and AVP stimulate the release of PG from different regions/cell types within the kidney.

Compartmental prostaglandin release by angiotensin II and arginine-vasopressin in rabbit isolated perfused kidneys.

The compartmental effects of angiotensin II (AII) and arginine-vasopressin (AVP) on renal prostaglandin (PG) formation were studied in the isolated perfused kidney of the rabbit by superfusion bioassay of venous and ureteral effluents (VE and UE) and radioimmunoassay (RIA). Comparable results were obtained with either bioassay or RIA when used to quantitate renal PG release. The effects on PG release into the VE were similar for AII and AVP, as were their pressor responses. However, their effects on PG release into the UE differed markedly. AII resulted in a 6-fold greater urinary efflux than venous of bioassayable PGs, whereas AVP-induced PG release into UE was slightly less than PG efflux into the VE at all doses of the peptide. The profile of released PGs varied according to the sampling source (VE or UE). Moreover, each peptide released a similar profile of PGs at all doses, i.e. UE PGE2 greater than PGF2 alpha greater than 6-keto PGF1 alpha; VE PGE2 greater than 6-keto PGF1 alpha greater than PGF2 alpha (TxB2 was not detected in either effluent). Thus, renal vascular PG release is similar for the vasoactive peptides, AII and AVP, whereas the urinary efflux of PGs is considerably greater in response to AII.