Basement Membrane Of Blood Vessels Research Articles

Developmental histology of the liver in the Balkan crested newt Triturus ivanbureschi (Caudata: Salamandridae)

The liver, a crucial organ for metabolic processes, has a generally uniform histological structure across amphibian taxa. However, ontogenetic changes, particularly those related to biphasic life cycle and metamorphosis, are less documented. Here, we explored and described the liver histology of an emerging model organism, the Balkan crested newt (Triturus ivanbureschi) at three ontogenetic stages: larval, juvenile (just after metamorphosis) and adult. At the larval stage, the liver is characterized by hepatocytes containing large lipid droplets, poorly developed basement membranes in the blood vessels, and a lack of melanin-rich macrophage centers. Juveniles show transitional characteristics between larvae and adults. Lipid droplets in hepatocytes are abundant, but also, at the juvenile stage the well-developed basement membrane of blood vessels and melanomacrophages are present, as in adults. The presence of lipid droplets in hepatocytes during larval and juvenile stages suggests the liver's role in fat storage and energy provision during development and growth. Melanomacrophages, which synthesize melanin, perform phagocytosis, and neutralize free radicals, have been found in juveniles (after metamorphosis) and increase with age. The biphasic life cycle and liver histology transition in Triturus newts provide an insight in changes in liver histology and make them a suitable model for studying fat deposition regulation and the evolution of the immune system in terrestrial vertebrates.

2.Cerebrovascular neuropathology in the brains of people with Down syndrome

AbstractBackgroundPeople with Down syndrome (DS) are at genetic risk of developing early‐onset Alzheimer’s disease (AD). By age 40 years, virtually all people with DS develop beta amyloid plaques and tau neurofibrillary tangles, typically followed by the development of AD dementia over a decade later. Despite having a low frequency of vascular risk factors (i.e., hypertension, atherosclerosis), people with DS show an age‐related progression of cerebral amyloid angiopathy (CAA), white matter hyperintensities, microbleeds, micro infarcts, and enlarged perivascular spaces by neuroimaging. Interestingly, the incidence of CAA at autopsy is significantly higher in people with DS than patients with late onset AD. Our main objective is to investigate the cerebrovascular pathology in DS autopsy cases.MethodFree floating 30 µm thick sections from the occipital cortex of DS cases with or without AD pathology (DSAD and DS, respectively, n = 5/group) were compared to age matched cognitively normal controls (n = 5). Sections were immunostained for collagen IV (basement membranes) and for fibrinogen (protein extravasation).ResultPreliminary results show that compared to controls, DSAD brains have impaired vascular integrity, observed as reduced collagen IV basement membrane labeling and evidence of protein extravasation which may reflect microbleeds observed using in vivo neuroimaging.ConclusionOur preliminary data suggest a disruption of blood vessel basement membranes observed as losses in collagen IV immunostaining in DSAD brains. Moreover, fibrinogen extravasation was consistently observed in DSAD brains. We are currently characterizing the age of onset of collagen IV disruption/losses and fibrinogen leakage into the brain parenchyma in the context of an AT(N) framework to understand when in AD progression, cerebrovascular pathology evolves in DS.

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DRUG DEVELOPMENT Reactions ShareShare onFacebookTwitterWechatLinked InRedditEmail C&EN, 2022, 100 (34), p 3September 26, 2022Cite this:C&EN 100, 34, 3Letters to the editorLessons from diabetes researchI found the article “Looking to Lysine” in the Aug. 29, 2022, issue (page 15) interesting and perplexing. I remember the days that pharmaceutical executives refused to consider “permanently bonding” drugs for fear of side effects. With a renewed interest in permanent bonding and lysine, I am surprised that the old diabetes literature has not been consulted.With respect to lysine and N-terminal amines (both reasonable nucleophiles), there is a large set of literature in the diabetes area. The aldehyde glucose is normal in the blood of people without diabetes and elevated in that of people with diabetes. Glucose will form a Schiff base with lysines and N-terminal amines (Biochem. Biophys. Res. Commun. 1975, DOI: 10.1016/0006-291X(75)90289-2). While this is reversible, in water and over time, the Schiff base undergoes an Amadori rearrangement, resulting in a stable ketoamine. This reaction is the source of the relatively well-known clinical assay for hemoglobin A1c to evaluate diabetic control (J. Clin. Invest. 1976, DOI: 10.1172/JCI108436). This reaction is also the source for other glycosylated proteins in people with diabetes (Pract. Diabetes Int.2000, 17, 21; Clin. Ophthalmol. 2021, DOI: 10.2147/OPTH.S300242), which cause some symptoms (for example, circulatory problems and vision problems caused by the glycosylation of blood vessel basement membranes and eye proteins). It seems this old information could help those moving forward to use amine nucleophiles as a method of designing permanently bonded drugs. Since one target seems to be cancer cells, it might even be interesting to do an epidemiological evaluation of people with diabetes and cancer (World J. Diabetes 2020, DOI: 10.4239/wjd.v11.i6.227).David N. HaneyLa Jolla, CaliforniaMeaning of sustainable growthI always cringe a little when I see the oxymoron sustainable growth appear in a news article. The most recent reference appeared in the subtitle on page 35 of the July 25, 2022, edition of C&EN: “Air pollution data are essential for sustainable growth.” As Rudy Baum used to say, perpetual growth is not possible on a finite planet. People that use this term should be obligated to describe the conditions under which growth can be sustainable. What does this mean? I equate the words sustainable growth with growth that can be maintained indefinitely (for example, till humans cease to exist as a species). In a science publication like C&EN, this term should not be allowed to be used glibly by a guest author without a foundation of facts to back it up.Jim SchulteCicero, Illinois

Notch signaling regulates vessel structure and function via Hspg2

The abnormal structure of tumor blood vessels is an important reason for the low efficacy of anti-tumor drugs. Notch signaling is an evolutionarily highly conserved signaling pathway that plays an important role in vessel development. However, the role and mechanism of Notch signaling in the formation of vascular structure is not fully understood. In this study, we demonstrated that blocking Notch signaling in endothelial cells (ECs) leads to obstructed tumor blood vessel basement membrane formation and the reduction of blood perfusion, as well as blood-retinal barrier (BRB) and blood-brain barrier (BBB) destruction in healthy mice. Endothelial Notch overactivation exacerbates the increases in tumor blood vessel basement membrane and blood perfusion ratio, and promotes recruitment of retinal vascular smooth muscle cells in neonatal mice. Notch signaling also regulates the formation of adhesion junctions (AJs) in ECs. In addition, we confirmed that Notch signaling regulates the AJs of ECs by regulating the expression of downstream gene Hspg2. This research is of great theoretical and practical significance for understanding the mechanism of tumor vascular structure formation as well as the search for new targets for vascular-targeted therapy.

Glymphatic System and Subsidiary Pathways Drive Nanoparticles Away from the Brain.

Although drug delivery systems (DDS) are efficient in brain delivery, they face failure in clinical settings due to their potential toxicity to the central nervous system. Little is known about where the DDS will go after brain delivery, and no specific elimination route that shares a passage with DDS has been verified. Hence, identifying harmless DDS for brain delivery and determining their fate there would strongly contribute to their clinical translation. In this study, we investigated nonreactive gold nanoclusters, which can deliver into the brain, to determine the elimination route of DDS. Subsequently, nanoclusters in the brain were systemically tracked and were found to be critically drained by the glymphatic system from the blood vessel basement membrane to periphery circulations (77.8 ± 23.2% and 43.7 ± 23.4% contribution). Furthermore, the nanoclusters could be actively transported across the blood-brain barrier (BBB) by exosomes (30.5 ± 27.3% and 29.2 ± 7.1% contribution). In addition, microglia promoted glymphatic drainage and passage across the BBB. The simultaneous work of the glymphatic system, BBB, and microglia revealed the fate of gold nanoclusters for brain delivery and provided a basis for further brain-delivery DDS.

INTESTINAL ANASTOMOTIC LEAK: HISTOLOGICAL AND IMMUNOHISTOCHEMICAL ASPECTS

Summary. The aim. Improving the results of treatment of patients with intestinal anastomotic leak by determining the role of undifferentiated connective tissue dysplasia in the development of these complications.
 Materials and methods The object of the study comprises 45 patients with anastomotic leak and with phenotypic signs of undifferentiated connective tissue dysplasia, who were treated in the Shalimov National Institute of Surgery and Transplantology during 2017-2020. Laboratory, histological, immunohistochemical studies and statistical analysis were performed.
 Results and discussion. With comprehensive study of tissue fragments small and large intestines revealed similar morphological characteristics in groups of phenotypic traits undifferentiated connective tissue dysplasia and with intestinal anastomotic leak. In immunohistochemical study of tissues with monoclonal antibodies to α-smooth muscle actin revealed uneven focal expression in smooth muscle cells and fibroblast; with monoclonal antibodies to Collagen IV there is a moderate positive expression in the basement membrane of blood vessels, in smooth muscle cells of the muscular layer of the vascular wall, in areas of connective tissue.
 Conclusions. Immunohistochemical examination of small and large intestinal tissues with monoclonal antibodies to Collagen IV and α-smooth muscle actin revealed signs of pathological connective tissue remodeling in the areas of anastomotic leak.

Distinct Fibroblast Lineages Give Rise to NG2+ Pericyte Populations in Mouse Skin Development and Repair.

We have examined the developmental origins of Ng2+ perivascular cell populations that adhere to the basement membrane of blood vessels, and their contribution to wound healing. Neural/glial antigen 2 (Ng2) labeled most perivascular cells (70–80%) in developing and adult mouse back skin, a higher proportion than expressed by other pericyte markers Tbx18, Nestin and Pdgfrβ. In adult mouse back skin Ng2+ perivascular cells could be categorized into 4 populations based on whether they expressed Pdgfrα and Pdgfrβ individually or in combination or were Pdgfr-negative. Lineage tracing demonstrated that although Ng2+ cells in embryonic and neonatal back skin contributed to multiple cell types they did not give rise to interfollicular fibroblasts within the dermis. Lineage tracing of distinct fibroblast populations during skin development showed that papillary fibroblasts (Lrig1+) gave rise to Ng2+ perivascular cells in the upper dermis, whilst Ng2+ perivascular cells in the lower dermis were primarily derived from reticular Dlk1+ fibroblasts. Following wounding of adult skin, Ng2+ dermal cells only give rise to Ng2+ blood vessel associated cells and did not contribute to other fibroblast lineages. The relative abundance of Ng2+ Pdgfrβ+ perivascular populations was comparable in wounded and non-wounded skin, indicating that perivascular heterogeneity was maintained during full thickness skin repair. In the wound bed Ng2+ perivascular populations were primarily derived from Lrig1+ papillary or Dlk1+ reticular fibroblast lineages, according to the location of the regenerating blood vessels. We conclude that Ng2+ perivascular cells represent a heterogeneous lineage restricted population that is primarily recruited from the papillary or reticular fibroblast lineages during tissue regeneration.

Рівні основного фактору росту фібробластів та сульфатованих глікозоаміногліканів в обміні сполучної тканини при пролапсі мітрального клапану та цукровому діабеті 1 типу

In order to study the influence of the main fibroblast growth factor on the indicators of the state of the main substance of the extracellular matrix, a comparative analysis of their levels in the blood serum was carried out in persons with mitral valve prolapse and type 1 diabetes mellitus. The data obtained showed that the highest level of FGF-2 was observed in patients with a combination of MVP and type 1 diabetes mellitus compared with isolated MVP and diabetes, the control group (p <0.01) Level II of the GAG ​​fraction, which is represented in the main substance of the extracellular the matrix of the heart valve apparatus was also the highest in comorbid pathology, significantly differing from the control group (p <0.05). The level of GAG fraction III was higher among diabetic patients, regardless of the presence or absence of MVP, which is associated with the localization of this GAG fraction in the basement membranes of blood vessels and may be one of the elements of the pathogenesis of diabetic angiopathy

Matrix Metalloproteinases MMP-2 and MMP-9 Occupy a New Role in Severe Preeclampsia.

Introduction Preeclampsia (PE) is a life-threatening condition for the mother, the fetus, and the newborn. Matrix metalloproteinases (MMP) participate in the two primary stages of PE: remodeling of blood vessels at the stage of placental formation and the development of hypertension due to damage to the basement membrane of blood vessels. The object of the present study was to reveal the role of MMP-2 and MMP-9 in the development of severe preeclampsia. Materials and Methods We conducted a retrospective study that included 92 pregnant women at a gestational age of 26-38 weeks, of which the principal group consisted of 61 patients with severe PE. We divided the principal group into two subgroups: the first subgroup was designated the severe early-onset preeclampsia (EO-PE) group and consisted of 30 pregnant women. The second group was designated the severe late-onset preeclampsia (LO-PE) group, comprising 31 patients. We determined the plasma concentrations of MMPs 2 and 9 in the groups with an ELISA. Results In the group of PE patients with both EO-PE and LO-PE, the level of MMP-2 was significantly higher compared to the women undergoing normal pregnancy; and we observed no significant differences when we compared the levels of MMP-2 in the subgroups with EO-PE and LO-PE. Analysis of the concentrations of MMP-9 in EO-PE and LO-PE subgroups revealed attenuated levels of MMP-9 in both groups relative to the control group. We also noted a diminished level of MMP-9 in the EO-PE group compared to the LO-PE group. Conclusions The significantly increased levels of MMP-2 in women—both in the EO-PE and LO severe PE subgroups—explain the participation of this enzyme in endothelial dysfunction in the second stage of severe PE. A diminution in MMP-9 in the EO-PE group confirmed the participation of MMP-9 in the process of spiral artery transformation.

Expression of collagen type IV in human kidney during prenatal development

Background/Aim. Type IV collagen belongs to the group of nonfibrillar collagens and is an important component of the basement membranes, where it accounts for approximately 50% of its structural elements. The aim of the study was to describe the expression and distribution of collagen type IV in the embryonic and fetal metanephric kidney and to determine the volume density of collagen type IV in kidney tissue in each trimester of development. Methods. The material consisted of 19 human embryos/fetuses, in the gestational age from 8th to 37th week. Kidney tissue specimens were routinely processed to paraffin molds, stained immunohistochemically using polyclonal anti-collagen IV antibody and counterstained with Mayer hematoxylin and eosin. Stained slides were examined using a light microscope, and images of the selected areas under different lens magnification were captured with a digital camera. Volume density of collagen type IV was determined using ImageJ 1.48v and a plugin of the software, which inserted a grid system with 336 points. For the data comparison, the One-Way Analysis of Variance (ANOVA) was used. Results. Strong collagen IV immunopositivity was seen in all specimens, with a distribution in the basement membranes of urinary bud, parietal leaf of Bowman?s capsule, glomerular basement membrane, basement membrane of interstitial blood vessels, and basement membranes of nephron tubules and collecting ducts. No statistically significant difference in the volume density of type IV collagen was found among the different trimesters of the embryonic and fetal development. Conclusion. The synthesis and secretion of collagen type IV simultaneously follow the development of nephron structures, collecting system and blood vessels. The volume density of collagen type IV remains constant throughout all the trimesters of metanephric kidney development, indicating that it plays a crucial role in the normal development of nephron and collecting system structures, as well as in maintaining the normal kidney function.

Genetic and morphological aspects of the enterocutaneous fistula development

Objective. To analyze the frequency of polymorphic variants of matrix metalloproteinase-2 (C-1306 → T) and tissue inhibitors of metalloproteinase-2 (G303 → A) genes in patients with enterocutaneous fistulas and to identify the connection with morphological changes of connective tissue.
 Materials and methods. The object of the study comprises 24 patients with enterocutaneous fistula who were treated in the Shalimov National Institute of Surgery and Transplantology during 2016-2020. Laboratory, genetic, histological studies and statistical analysis were performed.
 Results. As a result of genetic and statistical analysis of the matrix metalloproteinase-2 (C-1306→T) and tissue inhibitors of metalloproteinase-2 (G303→A) gene single nucleotide polymorphisms, genotype variants have been identified that are associated with the risk of enterocutaneous fistula development. Immunohistochemical examination of tissues with monoclonal antibodies to α-smooth muscle actin (α-SMA) revealed uneven, focal expression in smooth muscle differentiation cells and fibroblasts. Examination with monoclonal antibodies to Collagen IV there is a moderate positive expression in the basement membrane of blood vessels, in smooth muscle cells of the muscular layer of the vascular wall, in areas of connective tissue.
 Conclusion. Enterocutaneous fistula is 1,5 times more common in carriers of homozygous GG genotype of the tissue inhibitors of metalloproteinase-2 (G303→A) gene and twice less common in heterozygotes GA (25% vs. 40%, p=0,057). Carriers of minor homozygotes of AA genotype in the group with enterocutaneous fistula were not detected, while a similar genotype in the control group was found in 10% of cases. Immunohistochemical examination of small and large intestine tissues with monoclonal antibodies to Collagen IV and α-SMA revealed signs of pathological connective tissue remodeling.

Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is a biomarker of poor prognosis in primary melanomas, and its depletion inhibits melanoma cell invasion and disrupts tumor blood vessel walls.

Melanoma is an unpredictable, highly metastatic malignancy, and treatment of advanced melanoma remains challenging. Novel molecular markers based on the alterations in gene expression and the molecular pathways activated or deactivated during melanoma progression are needed for predicting the course of the disease already in primary tumors and for providing new targets for therapy. Here, we sought to identify genes whose expression in primary melanomas correlate with patient disease‐specific survival using global gene expression profiling. Many of the identified potential markers of poor prognosis were associated with the epithelial–mesenchymal transition, extracellular matrix formation, and angiogenesis. We studied further the significance of one of the genes, prolyl 4‐hydroxylase subunit alpha 1 (P4HA1), in melanoma progression. P4HA1 depletion in melanoma cells reduced cell adhesion, invasion, and viability in vitro. In melanoma xenograft assays, we found that P4HA1 knockdown reduced melanoma tumor invasion as well as the deposition of collagens, particularly type IV collagen, in the interstitial extracellular matrix and in the basement membranes of tumor blood vessels, leading to vessel wall rupture and hemorrhages. Further, P4HA1 knockdown reduced the secretion of collagen triple helix repeat containing 1 (CTHRC1), an important mediator of melanoma cell migration and invasion, in vitro and its deposition around tumor blood vessels in vivo. Taken together, P4HA1 is an interesting potential prognostic marker and therapeutic target in primary melanomas, influencing many aspects of melanoma tumor progression.

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL ANALYSIS OF CONNECTIVE TISSUE CHANGES IN PATIENTS WITH ENTEROCUTANEOUS FISTULA

We examined 43 patients with enterocutaneous fistula and connective tissue pathology, who were treated in the Shalimov National Institute of Surgery and Transplantology during 2017-2020.With comprehensive study of tissue fragments small and large intestines revealed similar morphological characteristics in groups of phenotypic traits undifferentiated connective tissue dysplasia and with external intestinal fistula. In immunohistochemical study of tissues with monoclonal antibodies to α-SMA in both groups revealed uneven focal expression in smooth muscle cells and fibroblast; with monoclonal antibodies to Collagen IV there is a moderate positive expression in the basement membrane of blood vessels, in smooth muscle cells of the muscular layer of the vascular wall, in areas of connective tissue, which are signs of pathological remodeling of connective tissue.

Heparanase: Historical Aspects and Future Perspectives.

Heparanase is an endo-β-glucuronidase that cleaves at a limited number of internal sites the glycosaminoglycan heparan sulfate (HS). Heparanase enzymatic activity was first reported in 1975 and by 1983 evidence was beginning to emerge that the enzyme was a facilitator of tumor metastasis by cleaving HS chains present in blood vessel basement membranes and, thereby, aiding the passage of tumor cells through blood vessel walls. Due to a range of technical difficulties, it took another 16years before heparanase was cloned and characterized in 1999 and a further 14years before the crystal structure of the enzyme was solved. Despite these substantial deficiencies, there was steady progress in our understanding of heparanase long before the enzyme was fully characterized. For example, it was found as early as 1984 that activated T cells upregulate heparanase expression, like metastatic tumor cells, and the enzyme aids the entry of T cells and other leukocytes into inflammatory sites. Furthermore, it was discovered in 1989 that heparanase releases pre-existing growth factors and cytokines associated with HS in the extracellular matrix (ECM), the liberated growth factors/cytokines enhancing angiogenesis and wound healing. There were also the first hints that heparanase may have functions other than enzymatic activity, in 1995 it being reported that under certain conditions the enzyme could act as a cell adhesion molecule. Also, in the same year PI-88 (Muparfostat), the first heparanase inhibitor to reach and successfully complete a Phase III clinical trial was patented.Nevertheless, the cloning of heparanase (also known as heparanase-1) in 1999 gave the field an enormous boost and some surprises. The biggest surprise was that there is only one heparanase encoding gene in the mammalian genome, despite earlier research, based on substrate specificity, suggesting that there are at least three different heparanases. This surprising conclusion has remained unchanged for the last 20years. It also became evident that heparanase is a family 79 glycoside hydrolase that is initially produced as a pro-enzyme that needs to be processed by proteases to form an enzymatically active heterodimer. A related molecule, heparanase-2, was also discovered that is enzymatically inactive but, remarkably, recently has been shown to inhibit heparanase-1 activity as well as acting as a tumor suppressor that counteracts many of the pro-tumor properties of heparanase-1.The early claim that heparanase plays a key role in tumor metastasis, angiogenesis and inflammation has been confirmed by many studies over the last 20years. In fact, heparanase expression is enhanced in all major cancer types, namely carcinomas, sarcomas, and hematological malignancies, and correlates with increased metastasis and poor prognosis. Also, there is mounting evidence that heparanase plays a central role in the induction of inflammation-associated cancers. The enzymatic activity of heparanase has also emerged in unexpected situations, such as in the spread of HS-binding viruses and in Type-1 diabetes where the destruction of intracellular HS in pancreatic insulin-producing beta cells precipitates diabetes. But the most extraordinary recent discoveries have been with the realization that heparanase can exert a range of biological activities that are independent of its enzymatic function, most notably activation of several signaling pathways and being a transcription factor that controls methylation of histone tails. Collectively, these data indicate that heparanase is a truly multifunctional protein that has the additional property of cleaving HS chains and releasing from ECM and cell surfaces hundreds of HS-binding proteins with a plethora of functional consequences. Clearly, there are many unique features of this intriguing molecule that still remain to be explored and are highlighted in this Chapter.

Blood–Brain Barrier Dysfunction After Smoke Inhalation Injury, With and Without Skin Burn

Only a handful of published reports exist today that describe neurological complications following smoke inhalation injury. In this study, we characterize acute pathophysiological changes in the brain of sheep exposed to smoke inhalation, with- and without third-degree skin burn that models the injuries sustained by human victims of fire accidents. Blood-brain barrier integrity and hemorrhage were analyzed throughout the brain using specific histological stains: Hematoxylin & Eosin, Luxol fast blue, Periodic acid-Schiff (PAS), and Martius, Scarlet and Blue (MSB). Our data show that, following smoke inhalation injury, alone and in combination with third-degree skin burn, there was a significant increase in the number of congested and dilated blood vessels in the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary gland as compared to sham-injured controls. Positive PAS staining confirmed damage to the basement membrane of congested and dilated blood vessels throughout the brain. Severe rupturing of blood vessels, microvascular hemorrhaging and bleeding throughout the brain was also observed in the injured groups. No significant changes in hemodynamics and PaO2 were observed. Our data demonstrate for the first time that acute smoke inhalation alone results in diffuse blood-brain barrier dysfunction and massive bleeding in the brain in the absence of hypoxia and changes in hemodynamics. These findings provide critical information and prompt further mechanistic and interventional studies necessary to develop effective and novel treatments aimed at alleviating CNS dysfunction in patients with smoke and burn injuries.

Cell signaling associated with internalization of 67 kDa laminin receptor (67LR) by soluble laminin and its implication for protection against neurodegenerative diseases.

Cell signaling associated with internalization of 67 kDa laminin receptor (67LR) by soluble laminin and its implication for protection against neurodegenerative diseases.

Anatomical and Histological Study of the Human Neonate Lungs

The current study aims to describe the normal anatomical structures of neonate lungs )weights, volumes and dimensions( as well as the architectural histology of structure In this study, 20 neonate cadavers collected with age ranging from 0-28 days, all these cadavers were collected from the Forensic Medicine in Baghdad and Kirkuk. Following dissection, the organ weight and gross metrical measurements were measured. Histological study was done on all cadavers enrolled in the study using Periodic Acid Schiff )PAS( Stain The results showed that the weight of neonatal lung was increased proportionally with age and the highest mean weight, volume and dimensions of lungs were found in neonates whose in the age group 22-28 day. The study revealed that dimensions of the left lungs were slightly more than those of right lungs especially in the age group 22-28 days. The study indicated that the volume of neonatal lungs were increased proportionally with age and the highest mean volume of lungs was found in neonates whose in the age group 22-28 days 50.03 cm3. The tunica media of the blood vessels of small and medium sized were well demonstrated of the thickness of alveolar walls also were obvious with inflammatory cell infiltration, the interstitial C.T. also associated with presence of smooth muscle cells, surrounded by segments of hyaline cartilage which are demonstrated easily, the lumen of bronchus appeared containing exudate, around the lung was containing with collagen bundle the elastic fibers. The basement membrane of the pulmonary blood vessels was demonstrated well by PAS which appeared thickened and the lumen of blood vessels was filled with blood serum and individual inflammatory cell. The interstitial C.T. around the blood vessels were containing dust cell with lymphocyte infiltration. .

Systemic Vascular Basement Membrane Markers Linked to Synovial Vascular Remodeling Are Bio-Markers of Hemarthrosis in Patients with Hemophilia

Introduction: Acute painful joint episodes in hemophilia are not always associated with hemarthrosis. Point-of-Care Musculoskeletal Ultrasound with Power Doppler (POC MSKUS/PD) is a fast and sensitive imaging modality to determine the presence of hemarthrosis and direct management. Systemic collagen cleavage products reflecting extracellular matrix, bone and soft tissue turnover may serve as an alternative tool for the detection of hemarthrosis. Based on previous observations of pronounced vascular remodeling within the synovial tissue during hemarthrosis in patients and hemophilic mouse models, we hypothesized that cleavage products of collagen IV, found in the basement membrane of blood vessels may differentiate between bleeding and non-bleeding states.Methods: Joint bleeding was induced in FVIII-/- mice (+/- FVIII-treatment before and shortly after injury). Synovial vascular remodeling was assessed 14 days later by MSKUS/PD and histology including αSmooth Muscle Actin (αSMA)-staining. Murine plasma samples were analyzed for C4M2 and P4NP-7S, which are type IV collagen turnover products. To determine human relevance, 31 adult patients with hemophilia (PWH), ≥ age 21, were studied prospectively for 3 years with MSKUS/PD during pain-free intervals and painful events for bleed status, vascular flow and 11 plasma markers of collagen turnover(C2M, ARGS, C3M, Pro-C3, C5M, Pro C5, C4M2, P4NP-7S, Col18N, DCN- CS, ColNeo10, NIC).Results: Type IV collagen turn-over markers were significantly elevated in plasma of FVIII-/- deficient mice 14 days after joint bleeding when compared to baseline (C4M2: 2.575 ng/mL vs. 1.94 ng/mL, p=0.029 and P4NP-7S: 80.3 ng/mL vs. 67.81 ng/mL, p= 0.0152 respectively ). This increase coincided with a significant increase in vessel number (2.3 fold, p=0.0008), vascular flow by PD (2.3 fold, p= 0.014) and αSMA staining (5.1 fold, p= 0.0007). C4M2 and P4NP7S plasma levels correlated negatively with αSMA staining, suggesting a role for αSMA in vessel stabilization (C4M2 p=0.02, Pearson r= -0.5707; P4NP-7S p= 0.0130, Pearson r= -0.6238). FVIII-treatment reduced plasma C4M2 and P4NP-7S levels to baseline (C4M2 1.98 ng/mL vs. 1.94 ng/mL, P4NP-7S 59.98 ng/mL vs. 67.81 ng/mL), but had no effect on the other vascular parameters. In patients, results from 91 visits were compiled. Twenty five were due to acute painful episodes with 16 confirmed hemarthroses. Of the 11 biomarkers evaluated, only C4M2 and P4NP-7S were transiently but significantly elevated in plasma during joint bleeding compared to pain free intervals or to non-bleeding painful episodes. Plasma C4M2 levels during acute painful bleeding episodes when compared to non-bleeding states were 41.65 vs. 29.69 ng/mL (p=0.0003), and P4NP-7S levels were 418.9 vs 278.1 ng/mL (p=0.0007), respectively. Baseline levels of C4M2 and P4NP-7S in PWH were above the lab reported normal range, indicating ongoing high vascular basement membrane turnover. Additionally, PWH who developed joint bleeding episodes had a higher level of collagen IV biomarkers at baseline when compared to PWH who did not experience joint bleeding (C4M2 33.91 vs 29.07 ng/mL, P4NP7s 320.6 vs 292.8 ng/mL), although this was not statistically significant. Hemarthrosis was accompanied by pronounced abnormal synovial microvascular flow evidenced by PD, while plasma levels of both biomarkers correlated positively with joint PD signal (C4M2, p= 0.0400, Pearson r=0.4409; P4NP7S-p=0.0141, Pearson r=0.5154).Conclusions: Our findings suggest that systemic vascular collagen turn-over products may be novel biomarker candidates to identify patients at high risk for joint bleeding and to diagnose acute hemarthrosis. Hemarthrosis in hemophilia is associated with pronounced synovial vascular remodeling. Cleavage products of type IV collagen, but not of any other collagen, were systemically elevated in PWH and distinguished bleeding from non-bleeding painful episodes. Type IV collagen is found exclusively in basement membranes and in the case of joint bleeding, the presumed source of these turnover markers is newly formed synovial blood vessels. Therefore, our findings support the concept that vascular instability during neovascularization is involved in the dynamics of hemophilic joint bleeding. These findings broaden the scope of potential diagnostic tools substantially, opening new avenues for personalized treatment of PWH. Disclosuresvon Drygalski:Bioverativ/Sanofi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria; HemaBiologics: Consultancy; Genentech: Consultancy; Biomarin: Consultancy, Honoraria; Uniqure: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Hematherix: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Ultrastructural Morphology of the Optic Nerve Head in Aged and Glaucomatous Mice.

PurposeTo study age- and intraocular pressure–induced changes in the glial lamina of the murine optic nerve on the ultrastructural level.MethodsNaïve C57bl/6 mice at various ages spanning the time between early adulthood (3 months) and senescence (30 months) were used in this study. In addition, the intraocular pressure (IOP) was increased in a group of young mice by injection of microbeads into the anterior chamber. The unmyelinated segments of the optic nerve containing the glial lamina were prepared for transmission electron microscopy and imaged at high resolution.ResultsAxon packing density decreased slightly with age. Aging nerves contained higher numbers of enlarged and degenerating axons. Mean axonal diameter and in particular the variance of axonal diameter correlated well with age. Axonal mitochondria also showed age-dependent signs of pathology. The mean diameter of axonal mitochondria increased, and aged axons often contained profiles of mitochondria with very few or no cristae. Astrocytic mitochondria remained normal even in very old nerves. Changes to axons and axonal mitochondria in young glaucomatous nerves were comparable with those of 18- to 30-month-old naïve mice. In addition to axons and mitochondria, aged and glaucomatous nerves showed thickening of the blood vessel basement membranes and increased deposition of basement membrane collagen.ConclusionsOn the ultrastructural level, the effects of age and elevated IOP are quite similar. One month of elevated IOP seems to have as strongly detrimental effects on the nerve as at least 18 months of normal aging.

Adaptive adhesion systems mediate glioma cell invasion in complex environments.

Diffuse brain invasion by glioma cells prevents effective surgical or molecular-targeted therapy and underlies a detrimental outcome. Migrating glioma cells are guided by complex anatomical brain structures but the exact mechanisms remain poorly defined. To identify adhesion receptor systems and matrix structures supporting glioma cell invasion into brain-like environments we used 2D and 3D organotypic invasion assays in combination with antibody-, peptide- and RNA-based interference. Combined interference with β1 and αV integrins abolished the migration of U-251 and E-98 glioma cells on reconstituted basement membrane; however, invasion into primary brain slices or 3D astrocyte-based scaffolds and migration on astrocyte-deposited matrix was only partly inhibited. Any residual invasion was supported by vascular structures, as well as laminin 511, a central constituent of basement membrane of brain blood vessels. Multi-targeted interference against β1, αV and α6 integrins expressed by U-251 and E-98 cells proved insufficient to achieve complete migration arrest. These data suggest that mechanocoupling by integrins is relatively resistant to antibody- or peptide-based targeting, and cooperates with additional, as yet unidentified adhesion systems in mediating glioma cell invasion in complex brain stroma.