Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.

Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.

PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation.

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

Pre-hatch lung development in the ostrich.

We studied development of the ostrich lung using light microscopy as well as electron microscopy techniques. At E24, the lung comprised a few epithelial tubes, interspersed with abundant mesenchyme with scattered profiles of incipient blood vessels. Between E24 and E39, the epithelial thickness was reduced by 90% from 13.5 ± 0.41 µm to 1.33 ± 0.014 µm (mean ± SD, respectively). Atria were evident at E32, and by E35, the first portions of the blood-gas barrier (BGB) measuring 3.41 ± 1.12 µm were encountered. Gas exchange tissue was well formed by E39 with atria, infundibulae, air capillaries and a mature blood-gas barrier (BGB). BGB formation proceeded through the complex processes of secarecytosis and peremerecytosis, which entailed decapitation of epithelial cells by cutting or pinching off respectively and by E39, the BGB was thin at 2.21 ± 1.21 µm. Vascular remodeling by intussusceptive angiogenesis was a late stage process mediated by intraluminal pillars in the pulmonary vasculature.

Intussusceptive angiogenesis: pillars against the blood flow.

Adaptation of vascular networks to functional demands needs vessel growth, vessel regression and vascular remodelling. Biomechanical forces resulting from blood flow play a key role in these processes. It is well-known that metabolic stimuli, mechanical forces and flow patterns can affect gene expression and remodelling of vascular networks in different ways. For instance, in the sprouting type of angiogenesis related to hypoxia, there is no blood flow in the rising capillary sprout. In contrast, it has been shown that an increase of wall shear stress initiates the splitting type of angiogenesis in skeletal muscle. Otherwise, during development, both sprouting and intussusception act in parallel in building the vascular network, although with differences in spatiotemporal distribution. Thereby, in addition to regulatory molecules, flow dynamics support the patterning and remodelling of the rising vascular tree. Herewith, we present an overview of angiogenic processes with respect to intussusceptive angiogenesis as related to local haemodynamics.

The pulmonary blood-gas barrier in the avian embryo: inauguration, development and refinement.

In vertebrates, efficient gas exchange depends primarily on establishment of a thin blood-gas barrier (BGB). The primordial air conduits of the developing avian lung are lined with a cuboidal epithelium that is ultimately converted to a squamous one that participates in the formation of the BGB. In the early stages, cells form intraluminal protrusions (aposomes) then transcellular double membranes separating the aposome from the basal part of the cell establish, unzip and sever the aposome from the cell. Additionally, better endowed cells squeeze out adjacent cells or such cells constrict spontaneously thus extruding the squeezed out aposome. Formation of vesicles or vacuoles below the aposome and fusion of such cavities with their neighboring cognates results in severing of the aposome. Augmentation of cavities and their subsequent fusion with the apical plasma membranes results in formation of numerous microfolds separating concavities on the apical part of the cell. Abscission of such microfolds results in a smooth squamous epithelium just before hatching.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody.

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Erratum in Int J Biol Markers 2007; 22 (4): 265-73. Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

Errata Corrige In the article 'Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma' by Velinov N et al, which was published in the October-December issue of the International Journal of Biological Markers (Int J Biol Markers 2007; 22 (4): 265-73), an author and his affiliation were omitted, namely G. Mishev. We reprint herewith the names of all authors together with their affiliations:N. Velinov1, D. Aebersold2*, N. Haeni1*, R. Hlushchuk1,4, G. Mishev1, F. Weinstein1, R. Sedlacek3, V. Djonov1,4 1 Institute of Anatomy, University of Bern, Bern 2 Department of Radiation Oncology, University of Bern, Inselspital, Bern - Switzerland 3 Institute of Biochemistry, University of Kiel, Kiel - Germany 4 Institute of Anatomy, University of Fribourg, Fribourg - Switzerland *The contribution of both authors was equivalent.

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Bryne's malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient''s outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

Microvascular endowment in the developing chicken embryo lung.

In the current study, the contribution of the major angiogenic mechanisms, sprouting and intussusception, to vascular development in the avian lung has been demonstrated. Sprouting guides the emerging vessels to form the primordial vascular plexus, which successively surrounds and encloses the parabronchi. Intussusceptive angiogenesis has an upsurge from embryonic day 15 (E15) and contributes to the remarkably rapid expansion of the capillary plexus. Increased blood flow stimulates formation of pillars (the archetype of intussusception) in rows, their subsequent fusion and concomitant delineation of slender, solitary vascular entities from the disorganized meshwork, thus crafting the organ-specific angioarchitecture. Morphometric investigations revealed that sprouting is preponderant in the early period of development with a peak at E15 but is subsequently supplanted by intussusceptive angiogenesis by the time of hatching. Quantitative RT-PCR revealed that moderate levels of basic FGF (bFGF) and VEGF-A were maintained during the sprouting phase while PDGF-B remained minimal. All three factors were elevated during the intussusceptive phase. Immunohistoreactivity for VEGF was mainly in the epithelial cells, whereas bFGF was confined to the stromal compartment. Temporospatial interplay between sprouting and intussusceptive angiogenesis fabricates a unique vascular angioarchitecture that contributes to the establishment of a highly efficient gas exchange system characteristic of the avian lung.

Matrix metalloproteinase-19 is a predictive marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

AIMS: To evaluate the expression of matrix metalloproteinase-19 (MMP-19) in oropharyngeal squamous cell carcinoma along with its association with structural features of invasiveness. To investigate whether MMP-19 expression correlates with lymphatic or systemic metastasis and prognosis in patients who have received definitive radiotherapy. METHODS AND RESULTS: The histological evaluation of the invasive front was based on Brynes malignancy grading system. We correlated the immunohistochemical expression pattern with morphological parameters which characterize tumor invasiveness such as keratinization, nuclear polymorphism, invasion pattern, and the host inflammatory response. Local immunoreactivity for MMP-19 was positively correlated with tumor invasiveness as reflected in its structural characteristics and the degree of nuclear polymorphism, and negatively correlated with the inflammatory response of the host. No correlation existed between MMP-19 expression and clinicopathological features (TNM stage, grade of differentiation) or a patient''s outcome and prognosis. CONCLUSIONS: This latter finding probably reflects the unique change for MMPs from high immunoreactivity within healthy tissue areas and non-invasive tumor parts, through absence in the least invasive neoplastic regions, to strong re-expression at a highly invasive front of the same tumor. Our findings indicate that MMP-19 can be used as a marker for tumor invasiveness in patients with oropharyngeal squamous cell carcinoma.

Epithelial transformations in the establishment of the blood-gas barrier in the developing chick embryo lung.

The tall epithelium of the developing chick embryo lung is converted to a squamous one, which participates in formation of the thin blood-gas barrier. We show that this conversion occurred through processes resembling exocrine secretion. Initially, cells formed intraluminal protrusions (aposomes), and then transcellular double membranes were established. Gaps between the membranes opened, thus, severing the aposome from the cell. Alternatively, aposomes were squeezed out by adjacent cells or were spontaneously constricted and extruded. As a third mechanism, formation and fusion of severed vesicles or vacuoles below the aposome and their fusion with the apicolateral plasma membrane resulted in severing of the aposome. The atria started to form by progressive epithelial attenuation and subsequent invasion of the surrounding mesenchyme at regions delineated by subepithelial alpha-smooth muscle actin-positive cells. Further epithelial attenuation was achieved by vacuolation; rupture of such vacuoles with resultant numerous microfolds and microvilli, which were abscised to accomplish a smooth squamous epithelium just before hatching.

Correlation between the tumoral expression of beta3-integrin and outcome in cervical cancer patients who had undergone radiotherapy.

Integrins are cell-surface receptors, which mediate cell-to-cell and cell-to-extracellular matrix adhesion. Besides playing an important role in tumour angiogenesis, beta3-integrin is also expressed in several types of epithelial cancer cells. It was the purpose of the present study to evaluate the prognostic value of beta3-integrin expression in patients with cervical cancer. Biopsies were taken from 82 patients with squamous cell or adenocarcinomas of the uterine cervix who had undergone external-beam radiotherapy with or without brachytherapy. These tissue samples were analysed immunohistochemically for the expression of beta3-integrin. The impact of immunoreactivity for beta3-integrin on survival end points was assessed by univariate and multivariate analyses, and its correlation with clinicopathological characteristics evaluated by crosstabulations. beta3-integrin was expressed in 61% (50 of 82) of the patients. Kaplan-Meier curves revealed local progression-free survival, distant metastasis-free survival and cause-specific survival to be significantly shorter (P-values according to the log-rank test: 0.002, 0.04 and 0.01, respectively) in patients with beta3-integrin expression. The prognostic impact of this parameter was even higher than for other well-known prognostic parameters and remained statistically significant in the multivariate analyses. beta3-integrin, which is expressed in the majority of patients with advanced cervical cancer, has a significant prognostic impact on outcome according to univariate and multivariate analyses.