Lucio Díaz-Flores

Pericytes as a supplementary source of osteoblasts in periosteal osteogenesis.

In the adult rat femur, lifting a periosteum strip with microscopic bone flakes on its deep surface, if performed without damaging the surrounding microcirculation, rapidly leads to new bone formation and angiogenesis. Using vascular labeling, the pericytes and endothelial cells (ECs) were labeled with monastral blue (MB) in the preformed, preexisting postcapillary venules of the periosteal microcirculation. MB was detectable by light and electron microscopy and it persisted in some of the daughter cells. Between one and 21 hours, the MB labeling was restricted to the pericytes and ECs of postcapillary venules. Immediately afterward, both pericytes and ECs of these vessels were activated and continued to show MB. The phenomenon of pericyte activation includes enlargement, disruption of their basal lamina, separation from the walls of the preformed vessels, and the presence of mitotic figures. At this stage, activated pericytes with MB in their cytoplasm, fibroblast-like cells, and transitional cell forms between them were seen in interstitial areas. After 27 hours, vascular buds appeared and MB was detected in some ECs and pericytes. Between three and six days, when bone-tissue development was observed, some osteoblasts were MB labeled. Previous findings support the hypothesis that when the periosteum is activated, the process of bone formation from cells already present in the periosteum is augmented by proliferation and differentiation of pericytes, which contribute a supplementary population of osteoprogenitor cells.

CD34+ stromal cells/fibroblasts/fibrocytes/telocytes as a tissue reserve and a principal source of mesenchymal cells.: Location, morphology, function and role in pathology

We review the morphofunctional characteristics of CD34+ stromal fibroblastic/fibrocytic cells (CD34+ SFCs) and report our observations. We consider the following aspects of CD34+ SFCs: A) The confusing terms applied to this cell type, often combining the prefix CD34 with numerous names, including fibroblasts, fibrocytes, dendrocytes, keratocytes, telocytes and stromal, dendritic, adventitial, supraadventitial, perivascular, paravascular and delimiting cells; B) Changes in their immunophenotype, e.g., loss of CD34 expression and gain of other markers, such as those defining mesenchymal and derivate cells (myofibroblasts, osteoblasts, chondroblasts, adipocytes); C) Morphology (elongated or triangular cell body and thin, moniliform, bipolar or multipolar cytoplasmic processes), immunohistochemistry (co-expression of and changes in molecular expression) and structure (characteristics of nucleus and cytoplasmic organelles, and points of contact and junctions in quiescent and activated stages by light and electron microscopy); D) Location and distribution in the vessels (adventitia or external layer), in the tissues (connective, adipose, blood, muscle and nervous) and in the organs and systems (skin, oral cavity and oropharynx, respiratory, digestive, urinary, male, female, endocrine and lymphoid systems, serosal and synovial membranes, heart, eye and meninges); E) Origin from the mesoderm and cranial neural crest in the embryo, and from stem cells (themselves or other cells) and/or peripheral blood pluripotent stem cells (circulating progenitor cells) in post-natal life; F) Functions, such as synthesis of different molecules, progenitor of mesenchymal cells, immunomodulation, parenchymal regulation (growth, maturation and differentiation of adjacent cells), induction of angiogenesis, scaffolding support of other cells and phagocytic properties. Since CD34+ SFCs are the main reservoir of tissue mesenchymal cells (great mesenchymal potential, probably higher than that proposed for pericytes and other stromal cells), we dedicate a broad section to explain their in vivo behaviour during proliferation and differentiation in different physiologic and pathologic conditions, in addition to their characteristics in the human tissues of origin (adult stem cell niches); G) Involvement in pathological processes, e.g., repair (regeneration and repair through granulation tissue), fibrosis, tumour stroma formation and possible CD34+ SFC-derived tumours (e.g., solitary fibrous tumour, dermatofibrosarcoma protuberans, giant cell fibroblastoma, nuchal-type fibroma, mammary and extramammary myofibroblastoma, spindle and pleomorphic cell lipoma, and elastofibroma) and H) Clinical and therapeutic implications.

Telocytes in neuromuscular spindles.

A new cell type named telocyte (TC) has recently been identified in various stromal tissues, including skeletal muscle interstitium. The aim of this study was to investigate by means of light (conventional and immunohistochemical procedures) and electron microscopy the presence of TCs in adult human neuromuscular spindles (NMSs) and lay the foundations for future research on their behaviour during human foetal development and in skeletal muscle pathology. A large number of TCs were observed in NMSs and were characterized ultrastructurally by very long, initially thin, moniliform prolongations (telopodes – Tps), in which thin segments (podomeres) alternated with dilations (podoms). TCs formed the innermost and (partially) the outermost layers of the external NMS capsule and the entire NMS internal capsule. In the latter, the Tps were organized in a dense network, which surrounded intrafusal striated muscle cells, nerve fibres and vessels, suggesting a passive and active role in controlling NMS activity, including their participation in cell‐to‐cell signalling. Immunohistochemically, TCs expressed vimentin, CD34 and occasionally c‐kit/CD117. In human foetus (22–23 weeks of gestational age), TCs and perineural cells formed a sheath, serving as an interconnection guide for the intrafusal structures. In pathological conditions, the number of CD34‐positive TCs increased in residual NMSs between infiltrative musculoaponeurotic fibromatosis and varied in NMSs surrounded by lymphocytic infiltrate in inflammatory myopathy. We conclude that TCs are numerous in NMSs (where striated muscle cells, nerves and vessels converge), which provide an ideal microanatomic structure for TC study.

Mechanical ventilation modulates TLR4 and IRAK-3 in a non-infectious, ventilator-induced lung injury model

BackgroundPrevious experimental studies have shown that injurious mechanical ventilation has a direct effect on pulmonary and systemic immune responses. How these responses are propagated or attenuated is a matter of speculation. The goal of this study was to determine the contribution of mechanical ventilation in the regulation of Toll-like receptor (TLR) signaling and interleukin-1 receptor associated kinase-3 (IRAK-3) during experimental ventilator-induced lung injury.MethodsProspective, randomized, controlled animal study using male, healthy adults Sprague-Dawley rats weighing 300-350 g. Animals were anesthetized and randomized to spontaneous breathing and to two different mechanical ventilation strategies for 4 hours: high tidal volume (VT) (20 ml/kg) and low VT (6 ml/kg). Histological evaluation, TLR2, TLR4, IRAK3 gene expression, IRAK-3 protein levels, inhibitory kappa B alpha (IκBα), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL6) gene expression in the lungs and TNF-α and IL-6 protein serum concentrations were analyzed.ResultsHigh VT mechanical ventilation for 4 hours was associated with a significant increase of TLR4 but not TLR2, a significant decrease of IRAK3 lung gene expression and protein levels, a significant decrease of IκBα, and a higher lung expression and serum concentrations of pro-inflammatory cytokines.ConclusionsThe current study supports an interaction between TLR4 and IRAK-3 signaling pathway for the over-expression and release of pro-inflammatory cytokines during ventilator-induced lung injury. Our study also suggests that injurious mechanical ventilation may elicit an immune response that is similar to that observed during infections.

Human resident CD34+ stromal cells/telocytes have progenitor capacity and are a source of αSMA+ cells during repair.

We studied the progenitor capacity of human resident CD34+ stromal cells/telocytes (SC/TCs) in the enteric wall affected by inflammatory/repair processes (appendicitis, diverticulitis of large bowel and Crohns disease of the terminal ileum) at different stages of evolution (inflammatory, proliferative and remodelling). In these conditions, CD34+ SC/TCs are activated, showing changes, which include the following overlapping events: 1) separation from adjacent structures (e.g., from vascular walls) and location in oedematous spaces, 2) morphological modifications (in cell shape and size) with presence of transitional cell forms between quiescent and activated CD34+ SC/TCs, 3) rapid proliferation and 4) loss of CD34 expression and gain of αSMA expression. These events mainly occur in the inflammatory and proliferative stages. During the loss of CD34 expression, the following findings are observed: a) irregular cell labelling intensity for anti-CD34, b) co-localization of CD34 and actin, c) concurrent irregular labelling intensity for αSMA and d) αSMA expression in all stromal cells, with total loss of CD34 expression. While CD34 expression was conserved, a high proliferative capacity (Ki-67 expression) was observed and vice versa. In the segments of the ileum affected by Crohns disease, the stromal cells around fissures were αSMA+ and, in the transitional zones with normal enteric wall, activated CD34+ SC/TCs were observed. In conclusion, human resident CD34+ SC/TCs in the enteric wall have progenitor capacity and are activated with or without differentiation into αSMA+ stromal cells during inflammatory/repair processes.

Lectin-Gold Localization of Fucose Residues in Human Gastric Mucosa

The oligosaccharides of the mucous gastric glycoproteins are involved in the protection of the gastric mucosa and are altered in different diseases. Therefore, it is important to know their composition in health, to better determine the alterations induced by the disease. Moreover, analysis of the molecular composition of the fundic gland cells has been previously used to obtain new insights into the origin of the different cell types. The aim of the present study was the localization in the subcellular structures of the fucose residues of the oligosaccharides in human fundic glands. For this, lectin cytochemical methods were used at the light and electron microscopic levels. They were combined with enzymatic and chemical treatments to characterize the nature of the oligosaccharide chains containing the fucose residues. The presence of this carbohydrate belonging to N- or O-linked oligosaccharides has been demonstrated in the secretory granules of the surface, gastric pit, mucous neck, and transitional cells of the fundic mucosa, and in the intracellular canaliculi and tubulovesicular system of the parietal cells. These fucose residues were added in the trans-Golgi regions to the elongating chains. Additional fucose linked to the innermmost N-acetylglucosamine of the N-linked oligosaccharides was found in the chief cells, being incorporated in the cis-Golgi. The findings in the transitional cells corroborate the origin of the chief cells from the mucous neck cells.

Perineurial cell tumor (perineurioma) with granular cells.

A form of benign cutaneous tumor with perineurioma findings and with the presence of associated granular cells is described. The two cases studied consisted of whorls made up of a high number of circumferentially arranged flattened cells, with perineurial characteristics, including bipolar cell processes, pinocytotic vesicles, a basal lamina, a positive immunoreactivity for EMA, and absence of immunostaining for S‐100 protein. The granular cells, enclosed within the whorls, contained densely packed vesicles, particles with an apparently solid core, as well as membrane‐limited vacuoles with disintegrating cellular organelles and electron‐dense amorphous material. While failing to demonstrate any immunoreactivity for EMA, the granular cells showed positivity for S‐100 protein, which supports their Schwann‐cell origin. Due to its morphological and immunohistochemical characteristics, this peculiar form of tumor can be considered as a perineurioma with perineurial cell whorls and granular cell changes occurring in associated Schwann cells at the center of the whorls.

Behaviour of telocytes during physiopathological activation.

We consider CD34+ stromal cells/telocytes (CD34+ SC/TCs) in normal and pathological conditions. These cells are involved in organisation and control of the extracellular matrix, structural support, creation of microenvironments, intercellular communication, neurotransmission, immunomodulation and immunosurveillance, inhibition of apoptosis, and control, regulation and source of other cell types. CD34+ SC/TCs are widely reported in the origin of interstitial cells of Cajal and in regeneration in the heart, skeletal muscle, skin, respiratory tree, liver, urinary system and the eye. In addition, we contribute CD34+ SC/TC hyperplasia associated with several processes, including neurogenous hyperplasia (neuroma of the appendix), hyperplasia of Leydig cells in undescended testes (Cryptorchidism), peripheral areas of inflammatory/repair processes (pericicatricial tissue and transitional zones between diseased segments in Crohns disease and normal bowel), benign tumours (neurofibromas, Antoni-B zones of neurilemmomas, granular cell tumours, and melanocytic nevi) and in some lesions with myxoid, oedematous and degenerative changes (Reinkes oedema, myxomatous mitral valve degeneration, thyroid-associated ophthalmopathy and basophilic degenerative changes of the collagen in the dermis). We pay particular attention to the role of CD34+ SC/TCs during repair through granulation tissue, including morphologic changes, loss of CD34 expression and gain of αSMA expression with myofibroblast transformation, and interactions with pericytes, endothelial and inflammatory cells. Finally, we consider CD34 or αSMA expression in stromal cells of malignant epithelial tumours, and the role of CD34+ SC/TCs in the origin of carcinoma-associated fibroblasts (CAFs) and myofibroblasts. In conclusion, CD34+ SC/TCs play an important role in the maintenance and modulation of tissue homeostasis and in morphogenesis/renewal/repair.

Uptake and intracytoplasmic storage of pigmented particles by human CD34+ stromal cells/telocytes: endocytic property of telocytes

We studied the phagocytic‐like capacity of human CD34+ stromal cells/telocytes (TCs). For this, we examined segments of the colon after injection of India ink to help surgeons localize lesions identified at endoscopy. Our results demonstrate that CD34+ TCs have endocytic properties (phagocytic‐like TCs: phTCs), with the capacity to uptake and store India ink particles. phTCs conserve the characteristics of TCs (long, thin, bipolar or multipolar, moniliform cytoplasmic processes/telopodes, with linear distribution of the pigment) and maintain their typical distribution. Likewise, they are easily distinguished from pigment‐loaded macrophages (CD68+ macrophages, with oval morphology and coarse granules of pigment clustered in their cytoplasm). A few c‐kit/CD117+ interstitial cells of Cajal also incorporate pigment and may conserve the phagocytic‐like property of their probable TC precursors. CD34+ stromal cells in other locations (skin and periodontal tissues) also have the phagocytic‐like capacity to uptake and store pigments (hemosiderin, some components of dental amalgam and melanin). This suggests a function of TCs in general, which may be related to the transfer of macromolecules in these cells. Our ultrastructural observation of melanin‐storing stromal cells with characteristics of TCs (telopodes with dichotomous branching pattern) favours this possibility. In conclusion, intestinal TCs have a phagocytic‐like property, a function that may be generalized to TCs in other locations. This function (the ability to internalize small particles), together with the capacity of these cells to release extracellular vesicles with macromolecules, could close the cellular bidirectional cooperative circle of informative exchange and intercellular interactions.

Choroid plexus papilloma with stromal deposition of amyloid and elastic material

Congophilic birefringent amyloid deposits, with immunostaining for transthyretin (TTR) and amyloid P, associated with numerous coarse, enlarged and thick elastic fibres, are reported in the stroma of two choroid plexus papillomas, a finding not previously described in choroid plexus tumours. TTR was expressed as aggregates of ‘doughnut-shaped’ bodies, in which the TTR-positive peripheral area encircled the elastic fibre (TTR-negative core). Ultrastructurally, the amyloid microfibrils surrounded the elastic fibres and appeared to continue into the microfibrillar mantle of the latter. The stromal TTR-amyloid deposits associated with abundant elastic fibres in tumours that occur in the choroid plexus may be related to the alteration (production/accumulation, insufficient breakdown and/or extracellular matrix modifications) of some of the choroid plexus functions (removal, target and source of polypeptides, including TTR synthesis) and may be of interest for future studies on choroid plexus polypeptide activity and on protein development into elastomeric and amyloidogenic microfibrils.