Protein composition and biomechanical properties of in vivo-derived basement membranes

Basement membranes (BMs) evolved together with the first metazoan species approximately 500 million years ago. Main functions of BMs are stabilizing epithelial cell layers and connecting different types of tissues to functional, multicellular organisms. Mutations of BM proteins from worms to humans are either embryonic lethal or result in severe diseases, including muscular dystrophy, blindness, deafness, kidney defects, cardio-vascular abnormalities or retinal and cortical malformations. In vivo-derived BMs are difficult to come by; they are very thin and sticky and, therefore, difficult to handle and probe. In addition, BMs are difficult to solubilize complicating their biochemical analysis. For these reasons, most of our knowledge of BM biology is based on studies of the BM-like extracellular matrix (ECM) of mouse yolk sac tumors or from studies of the lens capsule, an unusually thick BM. Recently, isolation procedures for a variety of BMs have been described, and new techniques have been developed to directly analyze the protein compositions, the biomechanical properties and the biological functions of BMs. New findings show that native BMs consist of approximately 20 proteins. BMs are four times thicker than previously recorded, and proteoglycans are mainly responsible to determine the thickness of BMs by binding large quantities of water to the matrix. The mechanical stiffness of BMs is similar to that of articular cartilage. In mice with mutation of BM proteins, the stiffness of BMs is often reduced. As a consequence, these BMs rupture due to mechanical instability explaining many of the pathological phenotypes. Finally, the morphology and protein composition of human BMs changes with age, thus BMs are dynamic in their structure, composition and biomechanical properties.     

Deficiency of a transmembrane prolyl 4-hydroxylase in the zebrafish leads to basement membrane defects and compromised kidney function

Prolyl 4-hydroxylases (P4Hs) catalyze the hydroxylation of collagens and hypoxia-inducible factor (HIF)-α subunits. We studied the zebrafish homologue of the recently characterized human transmembrane P4H (P4H-TM) that can hydroxylate HIF-α, but not collagens, in vitro and influence HIF-α levels in cellulo. The zebrafish P4H-TM mRNA had its highest expression in the eye and brain and lower levels in other tissues, including the kidney. Morpholino knockdown of P4H-TM in embryos resulted in a reduction in the size of the eye and head and morphological alterations in the head from 2 days postfertilization onward. In addition, pericardial edema, regarded as a sign of kidney dysfunction, developed from 3 days postfertilization onward. The phenotype was dependent on the P4H-TM catalytic activity because similar results were obtained with morpholinos targeting either translation initiation or catalytic residues of the enzyme. Structural and functional analyses of the morphant pronephric kidneys revealed fragmented glomerular basement membranes (BMs), disorganized podocyte foot processes, and severely compromised pronephric kidney function leading to proteinuria. The opacity of the eye lens was increased due to the presence of extra nuclei and deposits, and the structure of the lens capsule BM was altered. Our data suggest that P4H-TM catalytic activity is required for the proper development of the glomerular and lens capsule BMs. Many HIF target genes were induced in the P4H-TM-deficient morphants, but the observed phenotype is not likely to be mediated at least solely via the HIF pathway, and thus P4H-TM probably has additional, as yet unknown, substrates.     

Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney

Mice lacking exon 3 of perlecan (Hspg2) gene were generated by gene targeting. Exon deletion does not alter the expression or the reading frame but causes loss of attachment sites for three heparan sulfate (HS) side chains. Hspg2(Delta 3 / Delta 3) mice are viable and fertile but have small eyes. Apoptosis and leakage of cellular material through the lens capsule are observed in neonatal lenses, and lenses degenerate within 3 weeks of birth. Electron microscopy revealed altered structure of the lens capsule through which cells had formed extensions. No kidney malfunction, such as protein uria, was detected in Hspg2(Delta 3 / Delta 3) mutant mice, nor were ultrastructural changes observed in the glomerular basement membranes (BMs). To achieve further depletion in the HS content of the BMs, Hspg2(Delta 3 / Delta 3) mice were bred with collagen XVIII null mice. Lens defects were more severe in the newborn Col18a1(-/-) x Hspg2(Delta 3 / Delta 3) mice and degeneration proceeded faster than in Hspg2(Delta 3 / Delta 3) mice. The results suggest that in the lens capsule, HS chains have a structural function and are essential in the insulation of the lens from its environment and in regulation of incoming signals.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation.

Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation

Summary Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Shaping cells and organs in Drosophila by opposing roles of fat body-secreted Collagen IV and perlecan

Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in?vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, we used a GFP-based RNAi method (iGFPi) designed to knock down any GFP-trapped protein in Drosophila. We found with this method that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. We also show that incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. Our results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form.     

An ultrastructural analysis of isolated basement membranes in the acellular renal cortex: A comparative study of human and laboratory animals

Freshly harvested kidneys from New Zealand white rabbits, Sprague-Dawley white rats, rhesus monkeys, and transplant-quality human kidneys were used in this study. Minced renal cortical tissue blocks (<2 mm³) were treated with 1 mM EDTA, 3% Triton X-100, 0.025% DNAse, and 4% sodium deoxycholate in an effort to remove all cellular elements and leave the extracellular matrix (ECM) intact. These preparations showed remarkable structural preservation and all components of the ECM, including basement membranes (BMs), maintained their in vivo histoarchitectural relationships. By light microscopy, at least four major BM types were recognizable, including Bowman's capsular BM (BCBM), tubular BM (TBM), glomerular BM (GBM), and peritubular capillary BM (PTCBM). Scanning electron microscopy demonstrated that, despite the lack of supporting interstitium, GBMs in human, monkey, and rat (and rabbit to a lesser degree) exhibit intrinsic structural rigidity such that their convoluted spheroidal shapes are maintained following cell removal. Transmission electron microscopy showed that major BM types are morphologically heterogeneous and vary markedly within and between species. Randomized measurements showed that isolated BM thicknesses (lamina densa only) compared favorably with those reported in cellular preparations. Mean thicknesses of GBMs were within normal ranges in all species with or without power transformations to reduce right-sided skew of distribution curves. In all species, thickness of BCBM > TBM > GGBM > PTCBM. The striking morphologic heterogeneity of major BM types demonstrated in the acellular renal cortex is not surprising in view of recent biochemical analyses that show that BMs derived from different sources are compositionally disparate. We conclude that BMs should be evaluated and characterized individually and that morphologic definition of isolated BMs is necessary prior to further analysis.     

Structure and function of basement membranes

Basement membranes (BMs) are present in every tissue of the human body. All epithelium and endothelium is in direct association with BMs. BMs are a composite of several large glycoproteins and form an organized scaffold to provide structural support to the tissue and also offer functional input to modulate cellular function. While collagen I is the most abundant protein in the human body, type IV collagen is the most abundant protein in BMs. Matrigel is commonly used as surrogate for BMs in many experiments, but this is a tumor-derived BM-like material and does not contain all of the components that natural BMs possess. The structure of BMs and their functional role in tissues are unique and unlike any other class of proteins in the human body. Increasing evidence suggests that BMs are unique signal input devices that likely fine tune cellular function. Additionally, the resulting endothelial and epithelial heterogeneity in human body is a direct contribution of cell-matrix interaction facilitated by the diverse compositions of BMs.     

The Bi-Functional Organization of Human Basement Membranes

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.     

Immunofluorescence study of the formation of evolutionary stable lens proteins in chick embryos]

In the lens of fishes (carp, spiny dogfish) beta-crystallins were identified which were characteristic also of reptiles, amphibians, birds and mammals (evolutionary stable beta-crystallins). The dynamics of the formation of such beta-crystallins in 5--14 days old chick embryos was studied by the indirect immunofluorescence method with antisera to fish lens. These proteins are reliably indentified first at the lens sections from 7--8days old chick embryos. At all stages under study these beta-crystallins are localized mainly in the epithelial cells and practically not found in the lens fibers. They were, however, found in the fibrous (central) part of developing lens as well by the method of immunoelectrophoresis.     

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.     

The clinical application of 68Ga-PSMA PET/CT and regulating mechanism of PSMA expression in patients with brain metastases of lung cancer

Brain metastases (BMs) of lung cancer are common malignant intracranial tumours associated with severe neurological symptoms and an abysmal prognosis. Prostate-specific membrane antigen (PSMA) has been reported to express significantly in a variety of solid tumours. However, the clinical applications of ⁶⁸Ga-PSMA PET/CT and the mechanism of PSMA expression in patients with BMs of lung cancer have rarely been reported. Experiments with ⁶⁸Ga-PSMA PET/CT and immunohistochemical staining were conducted to evaluate the expression of PSMA from seven patients with BMs of lung cancer who accepted surgical treatment in Fudan University Shanghai Cancer Center between October 2020 and October 2021. The mechanism of PSMA expression in BMs of lung cancer was explored by using single-cell RNA sequencing. The median maximum standardized uptake value (SUVmax) in BMs was higher than that in primary lung cancer (8.6 ± 2.8 vs. 3.6 ± 1.3, P < 0.01). The mean SUVmax in BMs was 1.76-fold higher than that in the liver, which indicated the potential of PSMA radioligand therapy (PSMA-RLT) for BMs. BMs showed intense PSMA staining, while normal lung tissue had no PSMA staining and there was only faint primary lung cancer staining by immunohistochemistry (IHC). Single-cell RNA sequencing (scRNA-seq) analysis found that PSMA was mainly expressed in oligodendrocytes of BMs, whereas it was expressed at lower levels in solid cells of lung cancer. PSMA expression in oligodendrocytes might be regulated by the factors ATF3 and NR4A1, which were associated with ER stress.     

Preparation and anti-tumor efficiency evaluation of doxorubicin-loaded bacterial magnetosomes: magnetic nanoparticles as drug carriers isolated from Magnetospirillum gryphiswaldense

Bacterial magnetosomes (BMs) are commonly used as vehicles for certain enzymes, nucleic acids and antibodies, although they have never been considered drug carriers. To evaluate the clinical potential of BMs extracted from Magnetospirillum gryphiswaldense in cancer therapy, doxorubicin (DOX) was loaded onto the purified BMs at a ratio of 0.87 +/- 0.08 mg/mg using glutaraldehyde. The DOX-coupled BMs (DBMs) and BMs exhibited uniform sizes and morphology evaluated by TEM. The diameters of DBMs and BMs obtained by AFM were 71.02 +/- 6.73 and 34.93 +/- 8.24 nm, respectively. The DBMs released DOX slowly into serum and maintained at least 80% stability following 48 h of incubation. In vitro cytotoxic tests showed that the DBMs were cytotoxic to HL60 and EMT-6 cells, manifested as inhibition of cell proliferation and suppression in c-myc expression, consistent with DOX. These observations depicted in vitro antitumor property of DBMs similar to DOX. The approach of coupling DOX to magnetosomes may have clinical potential in anti-tumor drug delivery.     

Bioenergetic modulators hamper cancer cell viability and enhance response to chemotherapy

Gliomas are characterized by a marked glycolytic metabolism with a consequent production of massive amounts of lactate, even in the presence of normal levels of oxygen, associated to increased invasion capacity and to higher resistance to conventional treatment. This work aimed to understand how the metabolic modulation can influence tumour aggressive features and its potential to be used as complementary therapy. We assessed the effect of bioenergetic modulators (BMs) targeting different metabolic pathways in glioma cell characteristics. The in vivo effect of BMs was evaluated using the chicken chorioallantoic membrane model. Additionally, the effect of pre‐treatment with BMs in the response to the antitumour drug temozolomide (TMZ) was analysed in vitro. Cell treatment with the BMs induced a decrease in cell viability and in migratory/invasion abilities, as well as modifications in metabolic parameters (glucose, lactate and ATP) and increased the cytotoxicity of the conventional drug TMZ. Furthermore, all BMs decreased the tumour growth and the number of blood vessels in an in vivo model. Our results demonstrate that metabolic modulation has the potential to be used as therapy to decrease the aggressiveness of the tumours or to be combined with conventional drugs used in glioma treatment.     

Comparative study of the crystallin supramolecular structure in the carp, frog, and rat lenses by small-angle roentgen ray scattering

The supramolecular structure of crystallins in intact ocular lenses of carp, frog and rat as well as in the interior (nuclear) and outer (cortical) parts of these lenses was studied by the small-angle X-ray scattering method. The results show that the supramolecular structure of crystallins substantially varies both in lenses of different vertebrate species and in various parts of the same lens. In carp lens and in the cortical part of rat lens, crystallins have an ordered supramolecular structure, as indicated by a small-angle X-ray diffraction maximum in the region of Bragg distances 15-20 nm, whereas in frog lens and in the nuclear part of rat lens, the supramolecular structure of these proteins is disordered. The power-law X-ray scattering by rat lens nucleus may be evidence of fractal structures in the lens. A comparison of these results with literary data indicates that there is no obvious correlation between the type of supramolecular structure of crystallins and their polypeptide composition in lenses of different vertebrate species. The results suggest that the supramolecular ordering (short-range order) of crystallins is not a necessary condition for lens transparency.     

Transformation of blood monocytes to multinucleated giant cells in vitro: are there any differences between malignant and nonmalignant states?

Blood monocytes (BMs) from 139 subjects (70 malignant melanoma patients, 31 breast cancer patients, 38 healthy controls) were cultured for at least 7 days. The formation of multinucleated giant cells (MGCs), which was checked during the whole time of culture, was observed in all cases. By the seventh day MGCs represented 25-50% and during the second and third month more than 90% of all cells. Lymphokines and/or concanavalin A stimulation (16-34 cases respectively) of BMs was performed as well. This stimulation greatly accelerated MGC formation. There were no differences either in spontaneous or in stimulated fusion between the different groups compared.     

Comparative studies on aldose reductase from bovine, rat and human lens

A purification scheme for aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) developed using bovine lens tissue including an affinity chromatographic step is presented which is particularly suited for small quantities of lenses. Using the affinity chromatographic method as a key step also makes it possible to obtain preparations of rat lens aldose reductase which are homogeneous. The behavior of crude preparations of aldose reductase from human lens on both ion-exchange and affinity chromatography was similar to the chromatographic behavior of the enzyme from rat and bovine lens. Comparative studies of aldose reductase obtained from the lenses of the three species demonstrate the similarity of the enzymes. These comparisons were based on molecular weights, isoelectric points, chromatographic behavior and kinetic data. Homotropic cooperativity for both NADPH and glyceraldehyde, as evidenced by a downward curvature in the Lineweaver-Burk double-reciprocal plots, had been demonstrated for aldose reductase obtained from bovine lens (Sheaff, C.M. and Doughty, C.C. (1976) J. Biol. Chem. 251, 2696-2702). Similarly, cooperativity was observed with the enzyme from both rat and human lenses and the apparent Km values at both high and low concentrations of substrate are comparable for the lens aldose reductases from all three species for both substrates.     

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography.     

Lens fiber organization in four avian species: a scanning electron microscopic study

The three-dimensional organization of the eye lenses of the chicken, the canary, the song-thrush and the kestrel was studied using light and scanning electron microscopy. The lenses of birds are characterized by the presence of two distinct compartments: the annular pad and the main lens body, separated by a cavum lenticuli. The annular pad fibers had a hexagonal circumference all contained a round nucleus and except for the canary were smooth-surfaced and lacking anchoring devices. In the canary, however, the annular pad fibers were studded with edge protrusions and ball-and-socket junctions. The semicircular main lens body fibers of all four species were studded with ball-and-socket junctions and edge protrusions. In contrast with mammals these anchoring devices were present throughout the lens up to the embryonal nucleus. Superficially the main lens body fibers were extremely flat. Additionally membrane elevations and depressions and globular elements were found on these central fibers in three species, the kestrel being the exception. At the transition between annular pad and main lens body the fibers turned their course and the nuclei became oval and disappeared in the deeper aspect of the main lens body. The cavum lenticuli was filled with globules tied off from the annular pad fibers. It seems attractive to assume that the presence of a separated annular pad, a cavum lenticuli filled with globular elements, the extreme flatness of the superficial central fibers and the studding of these central fibers with anchoring devices up to the embryonal nucleus are morphological expressions of the mouldability of the bird's eye lenses and consequently would explain their efficient accommodative mechanism including formation of a lenticonus. The presence of nuclei in the annular pad fibers and their typical change at the transitional zone between annular pad and main lens body are suggestive for a two-phased differentiation in bird's lens fibers: differentiation of the germinative epithelial cells to annular pad fibers which migrate to the main lens body after which they differentiate further to main lens body fibers.