Anders Haegerstrand

Accumulation of T lymphocytes and expression of interleukin-2 receptors in nonrheumatic stenotic aortic valves

OBJECTIVES Cell-specific antibodies were used to identify immunocompetent cells in a comparison of valves from patients who had symptomatic tricuspid aortic stenosis with subjects who had no evidence of valvular heart disease. BACKGROUND Nonrheumatic valvular aortic stenosis is the most common valvular heart disease among adults. The biologic processes involved in the development of this disease are poorly understood. METHODS Tricuspid stenotic aortic valves were obtained from 19 patients undergoing surgery for nonrheumatic valvular aortic stenosis, and 10 control valves were collected at autopsy. The valves were fixed in formaldehyde, cryosectioned and stained with antibodies against fibroblasts, endothelial cells, macrophages, T lymphocytes and interleukin-2 receptors. A subset of valves were also analyzed with antibodies against T-helper cells and cytotoxic T cells. RESULTS Stenotic valves were characterized by a basal accumulation of calcium deposits and a cell-rich subendothelial thickening. The immunohistologic analysis indicated that the cells in the subendothelial connective tissue were fibroblasts. T lymphocytes appeared to be the most common cell type in the vicinity of the calcium deposits and were also found close to the endothelial lining of the valves. T-helper cells were more frequent than cytotoxic T cells. Expression of interleukin-2 receptors occurred at the same location as T lymphocytes. Control valves lacked subendothelial thickening and contained only few cells reacting with antibodies against lymphocytes and macrophages. CONCLUSIONS The presence of activated T lymphocytes in tricuspid stenotic valves suggests that immunologic mechanisms may be involved in the etiology of nonrheumatic aortic stenosis.

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

SummaryUsing antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissners corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

SummaryRat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallell decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.

Biologically modified LDL increases the adhesive properties of endothelial cells.

Adhesion of monocytes to the arterial endothelium is an important early event in atherosclerosis. Several lines of evidence have suggested that oxidation of low density lipoprotein (LDL) in the arterial wall may initiate the inflammatory-like process that generally is present in atherosclerotic lesions. In vitro, oxidation of LDL can be obtained both by exposure to divalent ions, such as Cu2+, or by incubation with different cell types, including monocytes and endothelial cells. The present study was designed to investigate the possible influence of oxidized LDL on the adhesive properties of endothelial cells. We report here that Cu(2+)-oxidized LDL is as effective as interleukin 1 beta in stimulating the ability of cultured human endothelial cells to bind U937 monocytic cells. The stimulation was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Biologically modified LDL, obtained by incubation with human peripheral blood monocytes, also enhanced the adhesiveness of endothelial cells. This effect was not due to an increased secretion of interleukin 1 beta from the monocytes exposed to LDL. Treatment of endothelial cells for 24 h with native LDL was also found to increase the adhesion of U937 cells. Exposure of endothelial cells to LDL for 24 h resulted in an oxidative modification of LDL. Furthermore, the antioxidant butylated hydroxytoluene inhibited both the endothelial-dependent oxidation of LDL as well as the increased adhesion of U937 cells, suggesting a coupling between these two processes. The results indicate that LDL, modified by exposure to monocytes or endothelial cells in the arterial wall, may increase the adhesive properties of the endothelium.

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin

SummaryCalcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin generelated immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.

Spinal axons in central nervous system scar tissue are closely related to laminin-immunoreactive astrocytes

Although transected central nervous system axons fail to regrow after injuries in adult mammals, they send sprouts into the scar tissue that forms at the lesion. We have investigated the relation between scar cells, laminin-like immunoreactivity and cut spinal axons in two previously characterized spinal cord lesion types. Labeling with antisera to glial fibrillary acidic protein and laminin demonstrated that the scar tissue formed after lesions in the rat and cat dorsal and ventral funiculi showed prominent gliosis and strong laminin-like immunoreactivity four days to one year postlesion. Axonal sprouts in the scar, visualized with antibodies to neurofilament (RT97) or by tracing using fluorescein-conjugated dextran, were ensheathed by a thin layer of strongly laminin-immunoreactive tissue. Immunoelectron microscopy demonstrated that axons in the scar were ensheathed predominantly by astrocytes, and that the surface of the cells outlining the axons in the scar showed strong laminin-like immunoreactivity. Adhesive and neurite orienting properties in the scar tissue were assessed in an in vitro system where PC12 cells were cultured on spinal cord slices from dorsal funiculus-lesioned rats. Very few cells adhered to the spinal cord section except for the part where the scar tissue had formed, where numerous cells were attached. The PC12 cells that had adhered to the scar tissue were mainly seen in parts of the scar that showed laminin-like immunoreactivity and their neurites predominantly followed tissue showing laminin-like immunoreactivity. The close association between axonal sprouts and laminin-like immunoreactivity indicates a role for laminin in axonal growth and/or guidance in the injured spinal cord.

Mononuclear leukocytes exposed to oxidized low density lipoprotein secrete a factor that stimulates endothelial cells to express adhesion molecules

In animals fed a hypercholesterolemic diet, development of atherosclerosis is preceded by attachment of mononuclear leukocytes to the arterial endothelium. Early lesions begin to develop as monocytes migrate into the intima and ingest lipids. A major part of these lipids is believed to be derived from oxidatively modified low density lipoprotein (LDL). In the present study we demonstrate that human mononuclear leukocytes exposed to low concentrations of copper-oxidized LDL secrete one or several factors that stimulate the expression of intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin-1, ELAM-1), whereas native LDL was found to be without effect. Exposure of endothelial cells to non-conditioned medium containing oxidized LDL did not influence the expression of adhesion molecules. Incubation of endothelial cells with conditioned medium from mononuclear cells grown in the presence of oxidized LDL also resulted in a three-fold increase in the binding of monocytoid U937 cells. The present findings suggest that mononuclear leukocytes exposed to oxidatively modified LDL in early atherosclerotic lesions may stimulate the recruitment of other leukocytes by secreting cytokines which induce the expression of adhesion molecules on the endothelium.

Serial cultivation of adult human endothelium from the great saphenous vein

The growth of endothelial cells from small segments of human great saphenous vein was investigated for possible functional studies of endothelial cell properties and endothelialization of cardiovascular prosthetic graft materials. Growth in medium containing fetal calf serum or human serum was compared, and the effects of adding compounds that increase intracellular cyclic adenosine monophosphate levels, that is, cholera toxin and isobutylmethylxanthine, were also examined. It was shown that human serum was more efficient in stimulating cell growth than fetal calf serum at all concentrations tested. It was also shown that the addition of cholera toxin and isobutylmethylxanthine significantly potentiated growth. Minimal Essential Medium with the addition of 40% human serum and cholera toxin (1 nmol/L) and isobutylmethylxanthine (33 mumol/L) was shown to be optimal. From a single segment (3 to 5 cm), 20 x 10(6) human saphenous vein endothelial cells corresponding to 2000 cm2 or more could be achieved after 3 to 4 weeks in culture. The human saphenous vein endothelial cell cultures retained their cobblestone appearance, expression of von Willebrand Factor (vWF)-antigen, and capacity for prostacyclin production after six passages. We suggest that this provides a practically useful method for studies of cultured endothelium and possibly for pre-endothelialization of cardiovascular prosthetic materials.

Effect of recombinant IGF binding protein-1 on primary cultures of human keratinocytes and fibroblasts : selective enhancement of IGF-1 but not IGF-2-induced cell proliferation

The present report describes the mitogenic effect of recombinant IGF-2 on cultured human keratinocytes and fibroblasts compared to that of IGF-1. Furthermore, the modulating effect of a recently expressed recombinant form of placental-derived IGF-binding protein 1 (IGFBP-1) on IGF-induced proliferation was examined. A dose-dependent increase, up to 100%, in cell proliferation was seen in cultured human keratinocytes with IGF-2 and -1 and the proliferative response was comparable to the effect of epidermal growth factor (EGF). In human fibroblasts, IGF-1 stimulated DNA synthesis up to 300% for IGF-1 and up to 200% for IGF-2. The mitogenic effect of IGF-1 was enhanced by IGFBP-1 in both cell types. In contrast, the IGF-2-induced mitogenic effect was unperturbed. These findings indicate that the interaction between IGFs and their binding proteins may induce different responses depending upon the ligand and the target cell.