E. Mandache

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal‐like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non‐conventional light microscopy (NCLM) on semi‐thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c‐kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi‐thin sections, ICLC represent about 1–1.5% of the atrial myocardial volume (vs.±45% working myocytes, ˜2% endothelial cells, 3–4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8–10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2–3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (˜420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.150±0.1 μm), very long for a non‐nervous cell (several tens of μm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was ˜65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c‐kit, variously co‐expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for α‐smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S‐100.

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC).

We have previously described interstitial Cajal‐like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non‐conventional light microscopy (Epon‐embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent ∼32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of μm), very thin (0.1‐0.5μm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and Mito Tracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil‐like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/1μm cell membrane length, or 2‐4% of the relative cytoplasmic volume, Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5‐10% and 1‐2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.

Interstitial Cajal‐like cells (ICLC) in myocardial sleeves of human pulmonary veins

We present here evidence for the existence of a new type of interstitial cell in human myocardial sleeves of pulmonary veins: interstitial Cajal‐like cell (ICLC). This cell fulfils the criteria for positive diagnosis of ICLC, including CD 117/c‐kit positivity. Transmission electron microscopy revealed typical ICLC with 2 or 3 very long processes (several tens of mm) suddenly emerging from the cellular body. Also, these processes appear moniliform but extremely thin (0.1–0.4 mm) under the resolving power of the usual microscopy. Cell processes establish close spatial relationships between each other, as well as with capillaries and nerve endings. ICLC appear located among the myocardial cells and particularly at the border between the myocardial sleeve and pulmonary vein wall.

Myocardial interstitial Cajal-like cells (ICLC) and their nanostructural relationships with intercalated discs: shed vesicles as intermediates

Intercalated discs (ID) are complex junctional units that connect cardiac myocytes mechanically and electrochemically. However, there is limited information concerning the cardiomyocyte interaction with interstitial non‐muscle cells. Our previous studies showed that myocardial interstitial Cajal‐like cells (ICLC) are located in between cardiomyocytes, blood capillaries and nerve fibres. Typically, ICLC have several very long, moniliform, cytoplasmic processes which establish closed contacts with nerve fibres, as well as each other. We report here ultrastructural evidence concerning the relationships of ICLC processes with ID. The ICLC cytoplasmic prolongations (tens micrometers length) preferentially pass by or stop nearby the ID. Transmission electron microscopy emphasized three distinct connecting features between the tips of ICLC extensions and myocytes at the ‘mouth’ of ID: free or budding shed vesicles, exocytotic multi‐vesicular bodies and direct contacts. In the last case, electron‐dense repetitive nanostructures (‘pillars’) (35–40 nm high and 100–150 nm wide, similar to adhesion molecules) fasten the ICLC to the myocytes. All these features suggest a juxtacrine and/or paracrine intercellular mutual modulation of ICLC and cardiomyocytes in the microenvironment of ID, possibly monitoring the cardiac functions, particularly the electrical activity.

Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling.

The human heart can be frequently affected by an organ‐limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long‐standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo‐red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10‐nm‐thick filaments were strictly extra‐cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.

Caveolae in smooth muscles: nanocontacts

Smooth muscle cell (SMC) caveolae have been investigated by quantitative and qualitative analysis of transmission electron microscopy (TEM) images of rat stomach, bladder and myometrium, guinea pig taenia coli, human ileum, and rat aortic SMCs. Ultrathin (below 30 nm) serial sections were used for examination of caveolar morphology and their connections with SMC organelles. Average caveolar diameter was smaller in vascular SMCs (70 nm, n=50) than in visceral SMCs (77 nm, n=100), but with the same morphology. Most of the caveolae, featured as flask‐shaped plasma membrane (PM) invaginations, opened to the extracellular space through a 20 nm stoma (21, 3nm) having a 7 nm thick diaphragm. A small percentage of caveolae (3%), gathered as grape‐like clusters, did not open directly to the extracellular space, but to irregular PM pockets having a 20‐30 nm opening to the extracellular space. In visceral SMCs, caveolae were disposed in 4 ‐ 6 rows, parallel to myofilaments, whilst aortic SMCs caveolae were arranged as clusters. This caveolar organization in rows or clusters minimizes the occupied volume, providing more space for the contractile machinery. The morphometric analysis of relative volumes (% of cell volume) showed that caveolae were more conspicuous in visceral than in vascular SMCs (myometrium ‐ 2.40%; bladder ‐ 3.66%, stomach ‐ 2.61%, aorta ‐ 1.43%). We also observed a higher number of caveolae per length unit of cell membrane in most visceral SMCs compared to vascular SMCs (myometrium ‐ 1.06/μm, bladder ‐ 0.74/μm, aorta ‐ 0.57/μm, stomach ‐ 0.48/μm). Caveolae increase the cellular perimeter up to 15% and enlarge the surface area of the plasma membrane about 80% in SMCs. Three‐dimensional reconstructions (15μ3) showed that most caveolae, in both visceral and vascular SMCs, have nanocontacts with SR (87%), or with mitochondria (10%), and only 3%, apparently, have no contact with these organelles. Usually, 15 nm wide junctional spaces exist between caveolae and SR, some of them with nanostructural links between each other or with mitochondria: direct contacts (space < 2 nm or none) and molecular links, so called ‘feet’ (about 12 nm electron dense structures between organellar membranes). Direct contacts possibly allow molecular translocation between the two membranes. Electron‐dense ‘feet’‐like structures suggest a molecular link between these organelles responsible for intracellular Ca2+ homeostasis (excitation‐contraction coupling or pharmaco‐mechan‐ical coupling). Close appositions (~15 nm) have also been observed between caveolae and perinuclear SR cisterna, suggesting that caveolae might be directly implicated in excitation‐transcription coupling.

Ultrastructural Defects of the Glomerular Basement Membranes Associated with Primary Glomerular Nephropathies

It is generally accepted that a glomerular basement membrane (GBM) thinner than 200 nm should be considered below normal. When this abnormality has a global and diffuse distribution, the associated clinical condition is a benign familial hematuria related to mutations of the COL4A4/COL4A3 genes, or an Alport syndrome. More often the GBM defects display a focal and segmental pattern, too small to express a thin glomerular basement membrane disease. The aim of this study is to emphasize statistical data concerning the pathogenic link between the renal glomerular diseases and the preexisting thin and very thin GBM. A series of 487 renal biopsies from adult patients has been thoroughly investigated both for nephropathologic diagnosis and the GBM ultrastructure. It has been statistically concluded that there is a close coexistence of primary glomerulonephritis and thin glomerular basement membranes with the role of a predisposing condition for immune complex deposition.

The predictive value of peritubular capillaries C3d deposition in IgA glomerulonephritis

The IgA glomerulonephritis (IgAGN) is one of the most common primary glomerulonephritis and has a variable and difficult to predict evolution toward the end‐stage nephrosclerosis. The deposition of C3d complement component in peritubular capillaries (PTCs) indicates a variant type of acute rejection while C3d deposition in primary glomerulonephritis (GN) is poorly documented. The aim of this study is to examine C3d expression in peritubular capillaries (PTCs) as a predictive marker and its correlation with the severity of renal injury in IgA glomerulonephritis. Polyclonal FITC conjugated rabbit anti‐human C3c and C3d antibodies were used for direct immunofluorescent evaluation of the C3c and C3d deposits in 24 kidney biopsies with IgA glomerulonephritis. The study revealed that the C3d deposits in peritubular capillaries were associated with known predictors for rapid progression of IgAGN: glomerular sclerosis (63.6%), atrophic tubules (90,9%) and interstitial sclerosis (81,8%). The intensity of the C3c glomerular immunofluorescent deposits was related with active lesions. Thus, the predictive value of C3d deposition on PTCs in IgAGN is worth to be taken into consideration as an unfavorable outcome of the disease and request further long run investigations.

Neoductular progenitor cells regenerate hepatocytes in severely damaged liver: a comparative ultrastructural study.

In severely injured liver, stem cells give rise to progeny that tend to replace lost hepatocytes. Neoductular reaction appears as an inherent stage of liver reconstruction following severe damage caused by different pathological mechanisms. Few ultrastructural types of progenitor cells have been described, and some molecular phenotypes of progenitor stages have been characterized, but the details of the differentiation process are largely unknown. We prepared for light and electron microscopy examination human liver from biopsies of patients with chronic active hepatitis, and rat liver with allyl alcohol‐induced periportal necrosis. We found that progenitor neoductular cells acquire the hepatocytic polarity pattern during a multi‐step process apparently involving cell migration and dissolution of neoductular basement membrane. An intermediate stage with “mixed” ductular and hepatocytic polarity was described.

Endothelial cell mitosis in experimental heart hypertrophy

Three groups of Wistar rats were subjected to swimming exercise for 1 h each day, 6 days per week. After 4 weeks of exercise, rats were killed and hearts, rendered hypertrophic by swimming exercise, were dissected and studied. Histoautoradiographic investigation suggested a highly significant proliferation of capillary wall cells in these hearts. A thorough ultrastructural investigation of the myocardium showed clear evidence of capillary neoformation. The growing capillaries first appeared as solid cords of pericyte-like cells in the interstitium. In the central area of these cords there were clefts lined by endothelial-like cells containing pinocytotic vesicles. These cells might be either transformed pericytes or, more probably, the result of endothelial proliferation. Such a mitotic endothelial cell, most probably in prophase, is shown in the center of the picture.