Macrophages of diverse phenotypes drive vascularization of engineered tissues.

Macrophages are key contributors to vascularization, but the mechanisms behind their actions are not understood. Here, we show that diverse macrophage phenotypes have distinct effects on endothelial cell behavior, with resulting effects on vascularization of engineered tissues. In Transwell coculture, proinflammatory M1 macrophages caused endothelial cells to up-regulate genes associated with sprouting angiogenesis, whereas prohealing (M2a), proremodeling (M2c), and anti-inflammatory (M2f) macrophages promoted up-regulation of genes associated with pericyte cell differentiation. In 3D tissue-engineered human blood vessel networks in vitro, short-term exposure (1 day) to M1 macrophages increased vessel formation, while long-term exposure (3 days) caused regression. When human tissue-engineered blood vessel networks were implanted into athymic mice, macrophages expressing markers of both M1 and M2 phenotypes wrapped around and bridged adjacent vessels and formed vessel-like structures themselves. Last, depletion of host macrophages inhibited remodeling of engineered vessels, infiltration of host vessels, and anastomosis with host vessels.

p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry.

In this study the direct involvement of cadherins in adhesion-mediated growth inhibition was investigated. It is shown here that overexpression of N-cadherin in CHO cells significantly suppresses their growth rate. Interaction of these cells and two additional fibroblastic lines with synthetic beads coated with N-cadherin ligands (recombinant N-cadherin ectodomain or specific antibodies) leads to growth arrest at the G1 phase of the cell cycle. The cadherin-reactive beads inhibit the entry into S phase and the reduction in the levels of cyclin-dependent kinase (cdk) inhibitors p21 and p27, following serum-stimulation of starved cells. In exponentially growing cells these beads induce G1 arrest accompanied by elevation in p27 only. We propose that cadherin-mediated signaling is involved in contact inhibition of growth by inducing cell cycle arrest at the G1 phase and elevation of p27 levels.

Cadherin-mediated transmembrane interactions.

We show in this study that cadherin ligands, either soluble or immobilized on different surfaces, can bind to cells carrying a compatible cadherin and induce long-range signals which affect cell adhesion and dynamics. Addition of recombinant N-cadherin extracellular domain (NEC) to CHO cells expressing N-cadherin (FL4) greatly enhanced the calcium-dependent aggregation of the cells and blocked their migration into an "in vitro wound". Monoclonal antibody which blocks cadherin interactions inhibited the aggregation of suspended FL4 cells and facilitated the "wound closure". As previously shown (Levenberg et al., 1998) synthetic beads coupled to NEC interacted specifically with the surface of FL4 cells and significantly enhanced the formation of adherens junctions. This effect was obtained also with the parental CHO cells, which contain low levels of N-cadherin, and in additional N-cadherin expressing cells such as cultured myoblasts. We further show here that stimulation of adhesion is not affected by the geometry of the NEC-bound surface and that cells plated on flat NEC-coated substratum also develop enhanced adherens junctions. Interaction of cells expressing low levels of endogenous N-cadherin, such as CHO cells with surface-immobilized N-cadherin ligands had a prominent effect also on the total level of N-cadherin and beta-catenin in the cells, probably due to stabilization of the cadherin-catenin complex by the interaction with the external surface.

Modulation of cell-cell adherens junctions by surface clustering of the N-cadherin cytoplasmic tail.

Cadherins mediate the formation of cell-cell adherens junctions (AJ) by homophilic interactions through their extracellular domains as well as by interacting with the actin cytoskeleton via their cytoplasmic portions. Cadherin clustering initiates cytoplasmic signaling that results in the assembly of structural components into cell-cell AJ. To elucidate the function of the cytoplasmic tail of cadherins in initiating the assembly signal, we generated and characterized a chimeric cadherin tail fused to an inert transmembrane anchor. The chimera enabled us to cluster the cadherin cytoplasmic tail in the absence of extracellular portions of the molecule. The transfected cadherin tail chimera localized to cell-cell AJ of epithelial cells, indicating that the submembrane junctional plaque has the capacity to recruit additional cadherins, with no involvement of their extracellular domains. Expression of the chimera in cells of mesenchymal origin resulted in dominant negative effects on the formation of cell-cell AJ. Surface clustering of cadherin cytoplasmic tails induced the recruitment of components and structural assembly of cell-cell AJ, thereby reversing the initial dominant-negative effects. We conclude that the cadherin cytoplasmic tail contains information required to direct the molecule to cell-cell AJ. Its function as modulator of cell-cell AJ depends on cell type and on whether the tail is clustered.

Long-range and selective autoregulation of cell-cell or cell-matrix adhesions by cadherin or integrin ligands.

In this study we demonstrate that local stimulation of cell surface cadherins or integrins induces a selective enhancement of adherens junction or focal contact assembly, respectively, throughout the cell. N-cadherin transfected CHO cells (CHO-Ncad) were incubated with different ligands including N-cadherin extracellular domain (NEC), anti-N-cadherin antibodies, fibronectin and concanavalin A (ConA), conjugated to synthetic beads. Electron microscopic examination indicated that both cadherin- and integrin-reactive beads bound tightly to the cell surface and were apparently endocytosed after several hours of incubation. The ConA-beads remained largely at the cell surface. Immunofluorescence labeling of the cells with antibodies to different adhesion-associated molecules indicated that both NEC- and anti-N-cadherin-conjugated beads induced a major increase in the level of junction-associated cadherin and beta-catenin labeling and a modest increase in junctional vinculin labeling, compared to untreated cells or cells bound to ConA-beads. FN-conjugated beads, on the other hand, significantly enhanced vinculin labeling at focal contacts and suppressed cadherin and beta-catenin staining in cell-cell junctions. The cadherin-reactive beads specifically stimulated tyrosine phosphorylation at cell-cell junctions, while the FN-beads increased the levels of focal contact-associated phosphotyrosine, as shown by immunofluorescence labeling of the cells for phosphotyrosine. Inhibition of this phosphorylation by genistein resulted in a complete suppression of the effects of both types of beads. These findings indicate that specific cadherin- and integrin-mediated surface interactions can trigger positively cooperative long-range signaling events which lead to the selective assembly of cell-cell or cell-matrix adhesions, and that these signals involve tyrosine phosphorylation.

Suppression of tumorigenicity by plakoglobin: an augmenting effect of N-cadherin.

Plakoglobin is a major component of the submembranal plaque of adherens junctions and desmosomes in mammalian cells. It is closely related to the Drosophila segment polarity gene armadillo which has a role in the transduction of transmembrane signals that regulate cell fate. Like its close homologue beta-catenin, plakoglobin can associate with the product of the tumor suppressor gene APC that is linked to human colon cancer. We have studied the effect of plakoglobin overexpression, and the cooperation between plakoglobin and N-cadherin, on the morphology and tumorigenic ability of cells either lacking, or expressing cadherin and alpha- and beta-catenin. Overexpression of plakoglobin in SV40-transformed 3T3 (SVT2) cells suppressed the tumorigenicity of the cells in syngeneic mice. Transfection with N-cadherin conferred an epithelial phenotype on the cell culture, but had no significant effect on the tumorigenicity of the cells. Cotransfection of plakoglobin and N-cadherin into SVT2 cells, however, was considerably more effective in tumor suppression than plakoglobin overexpression alone. Finally, transfection of plakoglobin into a human renal carcinoma cell line that expresses neither cadherins nor plakoglobin, or alpha-and beta-catenin, resulted in a dose-dependent suppression of tumor formation by these cells in nude mice. Plakoglobin, in these cells, did not exhibit junctional localization and was diffusely distributed in the cytoplasm, with a significant amount of the protein also localized in the nucleus. The results suggest that plakoglobin can efficiently suppress the tumorigenicity of cells in the presence of, or independently of the cadherin-catenin complex.

Molecular interactions in the submembrane plaque of cell-cell and cell-matrix adhesions.

Adhesion of cells to their neighbors or to the extracellular matrix has multiple effects on cell shape, dynamics and fate. The most obvious and direct one is the assembly of single cells into ordered multicellular tissues and organs. This process requires specific transmembrane adhesion molecules which mediate the binding to the external surface, cytoskeletal filaments which attach to the cytoplasmic faces of the adhesion site, and a submembrane plaque which interconnects the two. The co-assembly of these junctional domains is essential for the formation of stable cell adhesions with the proper mechanical properties. In addition, adhesive interactions have prominent, global consequences on cell behavior and fate, affecting such processes as differentiation, growth and survival. To gain insight into the molecular basis for both the local and global effects of adhesive interactions, we have chosen to focus on one specific junctional domain, the submembrane plaque of microfilament-bound adhesions, namely cell-cell and cell-matrix adherens junctions. Based on both biochemical and morphological evidence we would like to propose that the junctional plaque plays a key role in mediating and regulating transmembrane junctional interactions and adhesion-dependent signaling. It offers multiple modes of linkage between the cytoskeleton and the membrane, and its assembly can be controlled at either the biosynthetic or posttranslational levels. Furthermore, recent data demonstrate that the submembrane plaque is involved in the transduction of transmembrane signals. We will show that this structure is the residence of an array of signaling enzymes (mostly kinases), that its structure and composition may be affected by activation of various signaling systems, and that adhesion itself may activate specific signal transduction pathways.

Outpatient treatment of the problem drinker: strategies for attaining abstinence.

The initial phase of outpatient alcohol treatment is often the most difficult, since the patient must cease drinking--a change he has typically been unable to accomplish in the past. This paper describes a model of treatment which emphasizes that well-intentioned attempted solution behaviors by the patient and his family frequently serve to perpetuate the very drinking behavior they intend to eliminate. "Second order," illogical interventions can be a powerful clinical method for overcoming this vicious cycle of repetitive abusive drinking. The model is family oriented and conceptualizes cessation of drinking (in the motivated patient) as a product of undoing the commonsensical but ineffective attempts of the patient and his family. Two case studies are offered, illustrating the techniques.

Studies in family-oriented crisis intervention with hemodialysis patients.

This article describes a family-oriented crisis intervention approach to help patients with chronic renal failure adjust to the unique demands of home dialysis. In particular, home dialysis necessitates a working patient-dialysis partner relationship that has very adaptive problem solving skills. A couple whose permorbid relationship is dysfunctional will soon manifest this under the stress of home dialysis. The family-oriented therapist initiates only the minimal change necessary in the relationship to achieve successful dialysis. In the home training stage the premorbidly dysfunctional couple seems best treated in individual interviews, whereas premorbidly functional couples respond more favorably to conjoint interviews which capitalize on their underlying strengths. Couples in crisis who are dialyzing at home may require a highly structured, behaviorally-oriented contractual approach which includes all relevant family members. This "band-aid" approach temporarily reinstitutes successful dialysis while purchasing more time for the couple to develop new coping mechanisms. Finally, four case studies are presented, including one in which crisis intervention efforts failed.

Professional training, psychodiagnostic skill, and kinetic family drawings.

On the basis of Kinetic Family Drawings, doctoral level clinicians, predoctoral interns, and hospital secretaries judged 36 children to be normal or disturbed and indicated their degree of confidence in each rating. The groups were not found to differ in overall diagnostic accuracy or in degree of confidence, but professional training level did appear related to the ability to better a chance level of performance. The performance of a KFD expert was no better than the mean performance of judges in the three experimental groups.