Therapy of ulcus cruris of venous and mixed venous arterial origin with autologous, adult, native progenitor cells from subcutaneous adipose tissue: a prospective clinical pilot study.

BACKGROUND: The stromal vascular fraction (SVF) of adipose tissue consists of cellular subpopulations with distinct regenerative potential. OBJECTIVE: To investigate the regenerative capacities of autologous SVF cells in the treatment of chronic leg ulcers of venous (VLU) and arterial-venous (AVLU) origin. METHODS: Multimorbid ulcer patients received a singular topical treatment with 9-15 × 106 SVF cells, separated from abdominal lipoaspirates by digestion with collagenase and neutral protease and applied immediately after isolation. The primary endpoints were the change in wound size 12 weeks after treatment and evaluation of adverse events. Secondary endpoints included the time to complete wound epithelialization and change in pain levels. Postoperative wound treatment modalities and treatment of comorbidities were not intensified compared with pre-operative management. Follow-up period was at least 6 months. RESULTS: Sixteen elderly ulcer patients (seven with VLU, nine with AVLU) were treated as described. All VLU patients (median ulcer size: 48.25 cm2 ) and four of nine AVLU patients showed complete epithelialization of the ulcers within 71-174 days. In three patients with large ulcerations on both legs, ulcerations on the non-treated, contralateral leg also epithelialized. Patients reported a considerable rapid decrease in pain intensity by 2.5 points on average on a visual scale from 1 to 5 within the first 2 weeks after treatment. The patients were followed up for 9-44 months (median: 30 months). No severe side-effects were observed. CONCLUSIONS: The use of SVF cells presents an effective, minimally invasive option for the treatment of VLU and AVLU even in multimorbid patients. In patients with larger predominantly ischaemic AVLU and comorbidities, one-time application of the used amounts of SVF cells was not sufficient in the majority of cases.

Recurrent missense mutations in the hair keratin gene hHb6 in monilethrix.

Monilethrix is an autosomal dominant hair disorder characterized by a beaded appearance of the hair resulting from periodic thinning of the shaft (MIM 158000). The phenotype shows variable penetrance and results in hair fragility and patchy dystrophic alopecia. Mutations of the helix-encoded region in two hair-specific keratins (hHb1 and hHb6) have been identified as responsible for this disorder. We investigated two unrelated families from Russia and Colombia with monilethrix and found two missense mutations in hHb6. In the Russian family, we found a G to A transition at the first base of codon 402, resulting in a lysine substitution (GAG to AAG), designated E402K. In the Colombian family, affected patients carried a missense mutation of codon 413, involving a transition from G to A causing a lysine substitution (GAG to AAG), designated E413K. These two mutations have been identified in other monilethrix families from Europe. Our findings extend the body of evidence implicating recurrent hHb6 and hHb1 mutations in monilethrix families from around the world.

Atrichia caused by mutations in the vitamin D receptor gene is a phenocopy of generalized atrichia caused by mutations in the hairless gene.

Generalized atrichia with papules is a rare disorder characterized by loss of hair shortly after birth and development of cutaneous cysts. Mutations in the hairless gene (HR) cause this phenotype in both mouse and human. Here we present a case of atrichia with papules in a patient with a normal HAIRLESS gene but with mutations in both alleles of the VITAMIN D RECEPTOR. The patient exhibited vitamin D resistant rickets, which was confirmed by an absent response of her fibroblasts to 1,25-dihydroxyvitamin D3 in vitro. Similar to individuals with HAIRLESS mutations, her skin showed an absence of normal hair follicles and the presence of follicular remnants and cysts. The cyst epithelium contained keratin-15- and keratin-17-positive cells suggesting derivation from the hair follicle bulge and the presence of epithelial stem cells. Although hair loss has been reported in association with hereditary vitamin D resistant rickets, we now characterize this alopecia as clinically and pathologically indistinguishable from generalized atrichia with papules, which was previously thought to be caused only by mutations in HAIRLESS. These findings suggest that VDR and HR, which are both zinc finger proteins, may be in the same genetic pathway that controls postnatal cycling of the hair follicle.

Hairless is translocated to the nucleus via a novel bipartite nuclear localization signal and is associated with the nuclear matrix.

Hair follicle cycling is an exquisitely regulated and dynamic process consisting of phases of growth, regression and quiescence. The transitions between the phases are governed by a growing number of regulatory proteins, including transcription factors. The hairless (hr) gene encodes a putative transcription factor that is highly expressed in the skin, where it appears to be an essential regulator during the regression of the catagen hair follicle. In hairless mice, as well as humans with congenital atrichia, the absence of hr gene function initiates a premature and abnormal catagen due to a dysregulation of apoptosis and cell adhesion, and defects in the signaling required for hair follicle remodeling. Here, we report structure-function studies of the hairless gene product, in which we identify a novel bipartite nuclear localization signal (NLS) of the form KRA(X13) PKR. Deletion analysis of the mouse hr gene mapped the NLS to amino acid residues 409-427. Indirect immunofluorescence microscopy of cells transiently transfected with hairless-green fluorescent fusion proteins demonstrated that these amino acid residues are necessary and sufficient for nuclear localization. Furthermore, nuclear fractionation analysis revealed that the hr protein is associated with components of the nuclear matrix.

alphaB-crystallin interacts with cytoplasmic intermediate filament bundles during mitosis.

The small heat-shock protein alphaB-crystallin interacts with intermediate filament proteins. Using cosedimentation assay, we showed previously that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Furthermore, when NIH 3T3 cells were submitted to different stress conditions a dynamic reorganization of the intermediate filament network was observed concomitantly with the recruitment of alphaB-crystallins on the intermediate filament proteins. Thus, the intracellular state of alphaB-crystallin correlated directly with the remodeling of the intermediate filament network in response to stress. Here, we show data suggesting that alphaB-crystallin is implicated in remodeling of intermediate filaments during cell division. We investigated the intracellular distribution of alphaB-crystallin in naturally occurring mitotic NIH 3T3 cells and in neuroblastoma N2a and N1E115 cells. In NIH 3T3 cells, alphaB-crystallin remained diffused throughout the cell cycle. Subcellular fractionation of alphaB-crystallin showed that alphaB-crystallin remained in the cytosolic compartment during mitosis. Furthermore, alphaB-crystallin accumulated in mitotically arrested NIH 3T3 cells. This increased level of alphaB-crystallin protein was due to an increased level of alphaB-crystallin mRNA in mitotic NIH 3T3 cells. In the neuroblastoma cells, the intermediate filaments were rearranged into thick cable-like structures and alphaB-crystallin was recruited onto them. In neuroblastoma N2a cells the level of expression did not change during the cell cycle. However, a small fraction of alphaB-crystallin switched onto the insoluble fraction in mitotically arrested N2a cells. Our results suggested that depending on the state of rearrangement of the intermediate filament network during mitosis alphaB-crystallin was either recruited onto the intermediate filaments or upregulated in the cytosolic compartment.

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery.

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.

AlphaB-crystallin interacts with intermediate filaments in response to stress.

The small heat shock protein alphaB-crystallin interacts with intermediate filament proteins. Using a co-sedimentation assay, we showed that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Specifically, a synthetic peptide representing the first ten residues of alphaB-crystallin was involved in this interaction. When cells were submitted to different stress conditions such as serum starvation, hypertonic stress, or heat shock, we observed a dynamic reorganisation of the intermediate filament network, and concomitant recruitment of alphaB-crystallins on intermediate filament proteins. Under normal conditions alphaB-crystallin was extracted from cells by detergent. In stressed cells, alphaB-crystallin colocalised with intermediate filament proteins, and became resistant to detergent extraction. The intracellular state of alphaB-crystallin seemed to correlate directly with the remodelling of the intermediate filament network in response to stress. This suggested that alphaB-crystallin functions as a molecular chaperone for intermediate filament proteins.

Combined effects of GDNF, BDNF, and CNTF on motoneuron differentiation in vitro.

We have previously shown that glial cell line-derived neurotrophic factor (GDNF), in addition to promoting the survival of dopaminergic neurons in cultures from embryonic rat ventral mesencephalon,also increases the activity of choline acetyltransferase (ChAT) in the cranial motoneurons present in these cultures (Zurn et al.: Neuroreport 6:113-118, 1994). By using the intermediate filament protein peripherin as a motoneuron marker, we report here that GDNF increases the number of motoneurons as well as the length of their neurites. Brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) also promote ChAT activity, motoneuron survival, and neurite outgrowth in these cultures, but to varying degrees. Although these three molecules have similar effects on cultured motoneurons, we provide evidence for a distinct mode of action of GDNF, BDNF, and CNTF, since combinations of GDNF and BDNF, GDNF and CNTF, and BDNF and CNTF have either additive or synergistic effects on ChAT activity and motoneuron number. In addition to the previously described motoneuron-specific neurotrophic factors BDNF and CNTF, GDNF combined with the latter two factors may provide an important tool for the treatment of human motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy, both by increasing efficiency of treatment, and by decreasing the likelihood of deleterious side-effects.

Expression of peripherin in solid transplants of foetal spinal cord and dorsal root ganglia grafted to the injured cervical spinal cord of adult rats.

The expression of the neuronal type III intermediate filament protein peripherin was studied in E14 spinal cord fragments and E15 dorsal root ganglia 1-30 weeks after their transplantation to the injured cervical spinal cord of the adult rat. In the dorsal root ganglion transplants, the surviving neurons generally appeared as a rather healthy looking population of small strongly immunoreactive cells which are very similar to the small dorsal root ganglion neurons of adult control rats. In the spinal cord transplants, there were only a few peripherin-immunoreactive neurons, morphologically close to the motoneurons or to the preganglionic sympathetic neurons of adult rats. In both types of transplants, peripherin expression of the immunoreactive neurons was apparently correlated with the previously established ability of these transplanted neurons for extensive axonal growth into a co-grafted peripheral nerve.

Peripherin expression in hippocampal neurons induced by muscle soluble factor(s).

Previous studies have shown that neuronal cells in culture can switch neurotransmitters when grown in the presence of different target cells. To examine whether this plasticity extends to structural proteins, we cocultured hippocampal neurons and pituitary-derived neuroendocrine (AtT20) cells with astrocytes, kidney epithelial cells, or skeletal muscle cells. As a marker of phenotypic change we used the cytoskeletal protein peripherin, a type III intermediate filament (IF) subunit which is not expressed in hippocampal neurons and AtT20 cells. We show here that soluble factor(s) secreted specifically from skeletal muscle cells can induce the expression and de novo assembly of peripherin in a subset of post-mitotic neurons. We further demonstrate that one of these factors is the Leukemia Inhibitory Factor/Cholinergic Neuronal Differentiation Factor. The environmentally regulated expression of peripherin implies a remarkable degree of plasticity in the cytoskeletal organization of postmitotic CNS cells and provides a noninvasive model system to examine the de novo assembly of IF proteins under in vivo conditions.

Peripherin and neurofilaments: expression and role during neural development.

This review focuses attention on the expression of peripherin and the low-molecular mass neurofilament protein during development, as well as on recent results concerning the roles of these neuronal proteins. Peripherin is the only type III intermediate filament that has been shown to be expressed in neurons but exclusively in motor, sensory and sympathetic neurons; moreover, it is co-expressed with neurofilament proteins (NFP). Clearly, peripherin is expressed concomitantly with axonal growth during development, and its synthesis appears necessary to axonal regeneration in the adult. As to NFP, they are presumed to maintain the axonal diameter and thereby ensure a normal conduction velocity. In many neuropathies, either occurring in man or provoked by different means in animals, the neurofilament network is disrupted thus giving rise to bundles of filaments in perikarya or along axons; consequently, the axonal transport is impaired. The possible significance of the overexpression of NFP is discussed.

Structure of the mouse gene encoding peripherin: a neuronal intermediate filament protein.

The gene encoding mouse peripherin, a neuronal intermediate filament protein, has been cloned. Its sequence, through 1021 nucleotides composing the 5'-flanking region, nine exons, eight introns and 547 nucleotides of the 3'-flanking region, as well as its transcription initiation site have been determined. The amino acid coding sequence differs from that of the rat peripherin gene. The mouse gene has an additional histidine near the N-terminal end, and shows three conservative and two non-conservative changes. The promoter sequence, containing the binding sites for transcription factors as well as other sequences is homologous to promoter regions of other type III intermediate filament protein genes and other neuronal-specific genes.

Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments.

We studied the molecular associations between peripherin (a neuronal, type III intermediate filament subunit) and nuclear lamins. We show here that isolated peripherin binds selectively to mammalian lamin B under in vitro conditions. We further demonstrate that a synthetic peptide, representing the proximal part of peripherin's tail domain (P1), also associates with mammalian lamin B in a saturable, cooperative, and specific fashion. Laboratory animals immunized with P1 spontaneously develop idiotypic and anti-idiotypic antibodies recognizing peripherin and lamin B, respectively. These data provide essentially in vivo evidence that lamin B represents a constitutive nuclear "receptor" site for the tail domains of peripherin intermediate filaments.

Origin of the beta cells of the islets of Langerhans is further questioned by the expression of neuronal intermediate filament proteins, peripherin and NF-L, in the rat insulinoma RIN5F cell line.

Intermediate filament proteins of the rat insulinoma RIN5F cell line were characterized. Two-dimensional gel analysis followed by immunostaining of proteins demonstrated that these cells express both peripherin and the low-molecular-mass neurofilament protein (NF-L); this was confirmed for peripherin by immunohistochemistry, peptide analysis and Northern blot. No expression of these proteins could be detected with these same methods either in the adult pancreas or in the tumor at the origin of the cell line, although such expression was apparent on sections of rat pancreas at embryonal day 16. These results were compared to those obtained on the rat pheochromocytoma PC12 cell line: expression in the adrenal medulla of the embryo, no expression either in the adult tissue or in the tumor, but solely in the derived cell line. The expression of neuronal intermediate filament proteins in the rat insulinoma RIN5F cell line is discussed in relation to its similarity in the rat pheochromocytoma PC12 cell line, and its meaning as to the developmental cell lineage; an ectodermal origin is suggested for the pancreatic islet cells.

Differential expression of two neuronal intermediate-filament proteins, peripherin and the low-molecular-mass neurofilament protein (NF-L), during the development of the rat.

The expression of peripherin, an intermediate filament protein, had been shown by biochemical methods to be localized in the neurons of the PNS. Using immunohistochemical methods, we analyzed this expression more extensively during the development of the rat and compared it with that of the low-molecular-mass neurofilament protein (NF-L), which is expressed in every neuron of the CNS and PNS. The immunoreactivity of NF-L is first apparent at the 25-somite stage (about 11 d) in the ventral horn of the spinal medulla and in the posterior part of the rhombencephalon. The immunoreactivity of peripherin appears subsequently, first colocalized with that of NF-L. Both immunoreactivities then spread out along rostral and caudal directions, but whereas the immunoreactivity of NF-L finally becomes noticeable in every part of the nervous system, that of peripherin remains localized to (1) the motoneurons of the ventral horn of the spinal medulla; (2) the autonomic ganglionic and preganglionic neurons; and (3) the sensory neurons. These results demonstrate that, in the neurons that originate from migrating neural crest cells, the immunoreactivities of peripherin and of NF-L become apparent only when they have reached their destination. The results also show that peripherin is expressed more widely than has been previously observed and that this protein occurs in neuronal populations from different lineages (neural tube, neural crest, placodes) with different functions (motoneurons, sensory and autonomic neurons). The common point of these neurons is that they all have axons lying, at least partly, at the outside of the axis constituted by the encephalon and the spinal medulla; this suggests that peripherin might play a role in the recognition of the axonal pathway through the intermediary of membrane proteins.

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons.

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.

Adaptation to Pi deprivation of cell Na-dependent Pi uptake: a widespread process.

Phosphate enters kidney proximal tubular cells through an apical sodium-phosphate cotransport; this activity (Vmax) increases during phosphate deprivation (Kidney Int. 18: 36-47, 1980). This study investigated the mechanism of phosphate uptake and its adaptation to phosphate deprivation in cultured cells from different origins (kidney, LLC-PK1 and MDCK cells; liver, Fao cells; heart, myocyte primary cultures). All cells exhibited a sodium-dependent phosphate uptake that was reduced (greater than 75%) by external sodium substitution and inhibited by ouabain (35%) and 2,4-dinitrophenol or KCN (80%). Phosphate deprivation (exposure to phosphate-free medium) increased sodium-dependent phosphate uptake by 1.8- to 5.8-fold and decreased cell inorganic phosphate and ATP contents (70-80 and 17-30%, respectively). The stimulation of phosphate uptake resulted from an increase in Vmax without change in Km and was dependent on gene transcription and protein synthesis because it was inhibited by cycloheximide and 3-deoxyadenosine. Thus a deprivation-stimulated, sodium-dependent phosphate transport was demonstrated in cells originating from distal kidney tubules, liver, and heart. The findings suggest that in hypophosphatemic diseases, impairment of renal proximal phosphate reabsorption might be only one expression of a widespread alteration of cell phosphate regulation.