Omar Skalli

The Biology of the Myofibroblast Relationship to Wound Contraction and Fibrocontractive Diseases

The process of wound repair is of vital importance for animals as well as for plants (Shigo, 1985), since a wound perturbs body homeostasis and may result in infection by microorganisms. A wound may occur without or with tissue loss (Robbins et al, 1984). In both cases, but more clearly in the second case, wound healing consists schematically of acute inflammation followed by formation of granulation tissue, a transitional tissue able to retract the wound space, and finally scar formation.

Colonic pericryptal fibroblasts. Differentiation pattern in embryogenesis and phenotypic modulation in epithelial proliferative lesions.

Experimental evidence has shown that fetal gut mesenchymal cells can modulate epithelial cell differentiation. It is postulated that reciprocal stromal-epithelial interactions in the digestive tract are maintained beyond embryonic life. The mature colonic mucosa contains pericryptal fibroblasts (PCF), a stromal cell type exhibiting smooth muscle morphological features, which are thought to regulate the growth and differentiation of adjacent epithelial cells. Using an antibody directed at α-smooth muscle actin, which is constantly expressed in smooth muscle cells, we performed an immunohistochemical study on human embryonic tissues to assess PCF differentiation during development. PCF expressing ct-smooth muscle actin were first detected around the 21st week of gestation, at the bases of the crypts; the number of differentiated PCF increased then progressively, in synchrony with epithelial proliferation, to achieve at birth the characteristic distribution found in adults. We analyzed a series of non-malignant and malignant epithelial proliferative lesions of the adult colon by the same technique. Only sparse immunoreactive PCF were observed in 10/10 pure tubular adenomas, whereas in 11/11 villous adenomas immunoreactive PCF were consistently found bordering proliferative epithelia. Interestingly, 3/5 papillary adenomas, associated with areas of moderate to marked dysplasia, demonstrated foci of multi-layered immunoreactive PCF. In 14/14 carcinomas examined, PCF were no longer recognizable; stromal cells expressing variable amounts of β-smooth muscle actin, constituting the desmoplastic reaction, were constantly present. These observations establish immunohistochemically a smooth muscle phenotypic feature of PCF, which is acquired at mid-gestation, and the ability of PCF to proliferate in conjunction with some epithelial neoplasias. These findings might help to clarify the histogenesis of PCF and to improve our understanding of the mesenchymal-epithelial interactions suspected to operate during organogenesis as well as benign and malignant neoplastic conditions.

Alpha smooth muscle actin (α-SM actin) in normal human ovaries, in ovarian stromal hyperplasia and in ovarian neoplasms

SummaryAn immunohistochemical investigation of alpha-smooth muscle actin (α-SM actin) using the monoclonal anti-α-SM-1 antibody was carried out in 15 normal ovaries, in three ovaries with stromal hyperplasia and in 27 neoplastic ovaries. In selected cases the pattern of actin isoforms was examined by means of 2 D-gel electrophoresis. In addition, the tissues were stained for vimentin and desmin. In normal ovaries α-SM actin was found in the inner cortex and in the theca externa. In ovarian stromal hyperplasia expression of α-SM actin was minimal or absent. In primary and metastatic epithelial tumors there was positive stromal staining for α-SM actin, especially in the vicinity of epithelial elements. This tended to be more widespread in malignant neoplasms. Thecomas did not express α-SM-actin and could thus be differentiated from leiomyomas which stained intensely for α-SM actin. Only focal stromal staining of α-SM actin was observed in granulosa and germ cell tumors. In all the tissues studied blood vessels were strongly positive for α-SM actin. Desmin, although present in the stroma of most of the specimens, was less abundant than α-SM actin. We concluded that α-SM actin is a component of the normal human ovary where it may contribute to the contractility of its stroma. Its absence in the normal outer cortex and theca interna, and in stromal hyperplasia and thecoma implies that sex hormones do not constitute a stimulus for α-SM actin production in the ovary. Among neoplasms it is most widely represented in the stroma of epithelial tumors in which it may reflect stromal stimulation mediated by neoplastic epithelium.

α-Smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions

SummaryA series of 217 trephine bone marrow biopsies from adult patients and specimens from 16 fetuses and 5 infants were examined for the presence of stromal myoid cells (MCs) using a monoclonal antibody recognizing α-smooth muscle actin. In the normal adult bone marrow, stromal cells did not contain α-smooth muscle actin, whereas during fetal life, many α-smooth muscle actin-containing MCs were connected with vascular sinusoids in the primitive bone marrow. This cell type reappeared in various characteristic distribution patterns in adult bone marrow during different neoplastic and non-neoplastic conditions including metastatic carcinoma, Hodgkin’s disease, multiple myeloma, hairy cell leukemia, acute myeloid leukemia (FAB M4, 5, 7) and chronic myeloproliferative diseases. In general, the appearance of MCs was associated with a slight to pronounced increase in the deposition of reticulin and collagen fibers. We propose that bone marrow MCs represent a distinct subpopulation of fiber-associated or adventitial reticular cells undergoing cytoskeletal remodeling in response to various stimuli.

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

SummaryHuman arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed.

Distribution of cytoskeletal and contractile proteins in normal and tumour bearing salivary and lacrimal glands

We have evaluated by means of immunocytochemistry the distribution of various cytoskeletal and contractile proteins (cytokeratins, vimentin, desmin and α-smooth muscle actin) in 23 salivary or lacrimal gland primary tumours (15 pleomorphic adenomas and 8 carcinomas in pleomorphic adenoma), one third of which contained areas of normal gland. Normal epithelial luminal cells were stained by cytokeratin antibodies with a general specificity, while myoepithelial cells were selectively stained by a monoclonal antibody (SK2-27) reacting in immunoblots with cytokeratin polypeptides 14, 16 and 17, according to the classification of Moll et al. (1982) and by an antibody directed against α-smooth muscle actin (Skalli et al. 1986). In pleomorphic adenomas, both epithelial and myoepithelial cells displayed typical topographic distributions; moreover, myoepithelial cells showed two distinct cytoskeletal phenotypes. These findings could account in part for the heterogeneity of aspects observed in this tumour. In carcinomas, malignant cells were always positive to cytokeratin antibodies with general specificity and myoepithelial cells were absent as judged by anticytokeratin SK2-27 and anti-α-smooth muscle actin immunostainings. However, interestingly, there was in all cases a strong positivity for α-smooth muscle actin in stromal cells, similarly to what has previously been described for mammary carcinoma (Skalli et al. 1986). Our findings may be useful for the interpretation of the histogenesis of salivary and lacrimal tumour and stromal cells.

Action of general and α-smooth muscle-specific actin antibody microinjection on stress fibers of cultured smooth muscle cells

Arterial smooth muscle cells express alpha- and gamma-smooth muscle, as well as beta- and gamma-cytoplasmic actins. Two actin antibodies, one recognizing smooth muscle and cytoplasmic actin isoforms, the other recognizing specifically alpha-smooth muscle actin, were microinjected into cultured aortic smooth muscle cells. The effect of these antibodies on stress fiber organization was examined by staining with rhodamine-labeled phalloidin and by immunofluorescence with the same antibodies. Microinjection of the general actin antibody abolished most of the stress fiber staining with all reagents, but did not significantly affect the shape of the injected cells. This suggests that stress fiber integrity is not absolutely necessary for the maintenance of cell shape within the time of observation. Microinjection of the specific alpha-smooth muscle antibody abolished to various extents the staining of stress fibers with this antibody, but left practically intact their staining with rhodamine-labeled phalloidin and with the general actin antibody. This suggests that the incorporation of alpha-smooth muscle actin is not absolutely necessary for the maintenance of stress fiber integrity in cultured smooth muscle cells.

Expression, Organization, and Involvement of Intermediate Filaments in Disease Processes

Cytoskeletal and cytocontractile elements are involved in many cell physiological processes such as maintenance of cell shape, locomotion, mitosis, and secretion (for review see Alberts et. al., 1989). Moreover, the study of these structures may contribute to the understanding of cellular alteration during pathological situations (for review see Rungger-Brandle and Gabbiani, 1983). Different types of cytoskeletal changes may cause pathological situations. In some instances, a disease is due to the presence of an abnormal cytoskeletal protein. A well-documented example of this is the immotile cilia syndrome, characterized by an impaired mucociliary transport leading to chronic infections of airways (Camner et. al., 1975; Eliasson et. al., 1977); here, the basic defect is due to abnormal or missing dynein arms (Afzelius, 1985; Neustein et. al., 1980; Pedersen and Mygind, 1976). In other situations the primary defect affects a noncytoskeletal cellular component but leads to typical changes of the cytoarchitecture, as is the case for sickle-cell anemia where the primary defect is a mutant β-globin molecule leading to an altered organization of the cytoskeleton during the sickling process (Lux, 1979). In most instances, however, changes in the organization of cytoskeletal elements associated with pathological conditions reflect the degree of cellular adaptation to pathological stimuli (Rungger-Brandle and Gabbiani, 1983). Thus, cytoskeletal organization can be profoundly modified by pathological processes affecting cell activities such as migration or proliferation. Moreover, certain important pathological processes, such as tumor invasion, may at least in part depend on altered cytoskeletal organization (for review see Boschek, 1982).

Chemically induced rhabdomyosarcomas in rats

SummaryA series of 14 primary and two metastatic rat rhabdomyosarcomas (RMS) induced with nickel sulfide was studied by light microscopy, transmission electron microscopy, indirect immuno-fluorescence, avidin-biotin-peroxidase immuno-histochemistry and two-dimensional gel electrophoresis. Monoclonal or affinity-purified polyclonal antibodies were used for the immunohistochemical demonstration of vimentin, desmin, a-smooth muscle (α-sm) actin and α-sarcomeric (α-sr) actin. By histological and ultrastructural studies, four categories of RMS were diagnosed on the basis of the neoplastic cell types. These were: (1) well-differentiated RMS (n = 2), (2) pleomorphic RMS (n = 8), (3) embryonal RMS (n = 4), and (4) embryonal myosarcomas (n = 2). Immuno-histochemically, all these neoplasms expressed desmin and α-sr actin, reflecting their rhabdomyoblastic origin. Two dimensional gel electrophoresis performed on five neoplasms demonstrated α, β and γ actins spots in all cases. This study demonstrates that the α-sr actin antibody represents a good marker for rhabdomyoblastic differentiation is useful in the diagnosis of RMS since it was present in all morphologically confirmed RMS and in two ultrastructurally undifferentiated sarcomas positive for desmin. Neoplastic cells positive for α-sm actin were noted in 11 confirmed RMS. Double indirect immunofluorescence showed that all α-sm and α-sr positive cells also contained desmin. Co-expression of α-sr and α-sm actins was studied in serial sections of formalinfixed, paraffin-embedded tumor tissue. Both α-sm and α-sr actins were localized in some rhabdomyoblasts. This study confirms our previous observations in human tumors and shows, for the first time, that α-sr and α-sm actins can be present in the same neoplastic cell in vivo.