Moritz A. Konerding

Angiogenesis Inhibitors Endostatin or TNP-470 Reduce Intimal Neovascularization and Plaque Growth in Apolipoprotein E–Deficient Mice

BACKGROUND Neovascularization within the intima of human atherosclerotic lesions is well described, but its role in the progression of atherosclerosis is unknown. In this report, we first demonstrate that intimal vessels occur in advanced lesions of apolipoprotein E-deficient (apoE -/-) mice. To test the hypothesis that intimal vessels promote atherosclerosis, we investigated the effect of angiogenesis inhibitors on plaque growth in apoE -/- mice. METHODS AND RESULTS ApoE -/- mice were fed a 0.15% cholesterol diet. At age 20 weeks, mice were divided into 3 groups and treated for 16 weeks as follows: group 1, recombinant mouse endostatin, 20 mg. kg-1. d-1; group 2, fumagillin analogue TNP-470, 30 mg/kg every other day; and group 3, control animals that received a similar volume of buffer. Average cholesterol levels were similar in all groups. Plaque areas were quantified at the aortic origin. Median plaque area before treatment was 0.250 mm2 (range, 0.170 to 0.348; n=10). Median plaque areas were 0.321 (0.238 to 0.412; n=10), 0.402 (0.248 to 0.533; n=15), and 0.751 mm2 (0.503 to 0.838; n=12) for the endostatin, TNP-470, and control groups, respectively (P</=0.0001). Therefore, endostatin and TNP-470 inhibited plaque growth during the treatment period by 85% and 70%. Intimal smooth muscle cell contents of plaques from control and treated mice were similar. CONCLUSIONS Prolonged treatment with either angiogenesis inhibitor reduced plaque growth and intimal neovascularization in apoE -/- mice. Although the mechanism of plaque inhibition induced by these agents is not established, these results suggest that intimal neovascularization may promote plaque development.

Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells

Proteolysis mediated by the ubiquitin‐proteasome system has been implicated in the regulation of programmed cell death. Here we investigated the differential effects of proteasomal inhibitors on the viability of proliferating and quiescent primary endothelial cells in vitro and in vivo. Subconfluent, proliferating cells underwent carbobenzoxy‐L‐isoleucyl‐7‐í‐butyl‐L‐glutamyl‐L‐alanyl‐L‐leucinal (PSI)‐induced apoptosis at low concentrations (EC50=24 nM), whereas at least 340‐fold higher concentrations of PSI were necessary to obtain the same effect in confluent, contact‐inhibited cells. PSI‐mediated cell death could be blocked by a caspase‐3 inhibitor (Ac‐DEVD‐H), but not by a caspase‐1 inhibitor (Ac‐YVAD‐H), suggesting that a caspase‐3‐like enzyme is activated during PSI‐induced apoptosis. When applied to the embryonic chick chorioallantoic membrane, a rapidly expanding tissue, PSI induced massive apoptosis also in vivo. PSI treatment of the CAM led to the formation of areas devoid of blood flow due to the induction of apoptosis in endothelial and other cells and to the collapse of capillaries and first order vessels. Our results demonstrate that proteasomal inhibitors such as PSI may prove effective as novel anti‐angiogenic and anti‐neoplastic substances.—Drexler, H. C. A., Risau, W., Konerding, M. A. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. FASEB J. 14, 65–77(2000)

Liposuction-assisted nerve-sparing extended radical hysterectomy : Oncologic rationale, surgical anatomy, and feasibility study

OBJECTIVE Our purpose was to improve the therapeutic index of radical hysterectomy by extending the resection of parametrial tissue without further impairing pelvic autonomic nerve functions. STUDY DESIGN We studied the topographic anatomy of the parametrial tissue with high-resolution magnetic resonance imaging and by dissection of fresh human cadavers. We then performed a clinical feasibility study of the liposuction-assisted nerve-sparing extended radical hysterectomy. RESULTS Magnetic resonance imaging demonstrated that the perispinous adipose tissue is retained after type III radical hysterectomy and is a frequent site of tumor recurrence. The perispinous adipose tissue contains the pelvic plexus, the pelvic splanchnic nerves, small blood vessels, and lymphatic tissue. We developed the liposuction-assisted nerve-sparing extended radical hysterectomy and applied it to seven consecutive patients with cervical or vaginal cancer. No intraoperative or postoperative complications occurred. Postoperative magnetic resonance imaging assured us that perispinous adipose tissue was cleared in all cases. A metastatic lymph node was found in the perispinous adipose tissue removed by liposuction from one patient. Suprapubic cystostomies could be removed after a median period of 12 days. CONCLUSION The nerve-sparing removal of perispinous adipose tissue by liposuction is a feasible addition to wide en bloc parametrectomy in anatomically defined planes.

Tensile forces stimulate vascular remodeling and epidermal cell proliferation in living skin.

Objectives:To quantify tissue remodeling induced by static and cyclical application of tensional forces in a living perfused tissue. Background:Cells are able to respond to mechanical cues from the environment and can switch between proliferation and quiescence. However, the effects of different regimens of tension on living, perfused skin have not been characterized. Methods:The ears of living rats were mechanically loaded by applying tensile forces (0.5 Newtons) either statically or cyclically and then analyzing tissue responses using in vivo microscopy, immunohistochemistry, and corrosion casting. Results:Quantitative immunohistochemistry showed that in the static group (4-day continuous tension) there was up to 4-fold increase in cellular proliferation in the epidermis after 4 days and a 2.8-fold increase in the vascularity in the dermis that peaked after 2 days. Comparable effects could be achieved in just 8 hours using a cyclic loading protocol. We also modeled the resultant stress produced in the ear using a linear finite element model and demonstrated a correlation between the level of applied stress and both epidermal cell proliferation and blood vessel density. Conclusions:Mechanical forces stimulate cell proliferation and vascular remodeling in living skin. As cell growth and vascular supply are critical to wound healing and tissue expansion, devices applying controlled mechanical loads to tissues may be a powerful therapy to treat tissue defects.

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy

BACKGROUND Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiological function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Resection of the mesopancreas (RMP): a new surgical classification of a known anatomical space

BackgroundPrognosis after surgical therapy for pancreatic cancer is poor and has been attributed to early lymph node involvement as well as to a strong tendency of cancer cells to infiltrate into the retropancreatic tissue and to spread along the peripancreatic neural plexuses. The objective of our study was to classify the anatomical-surgical layer of the mesopancreas and to describe the surgical principles relevant for resection of the mesopancreas (RMP). Immunohistochemical investigation of the mesopancreatic-perineural lymphogenic structures was carried out with the purpose of identifying possible routes of metastatic spread.MethodsResection of the mesopancreas (RMP) was performed in fresh corpses. Pancreas and mesopancreas were separated from each other and the mesopancreas was immunohistochemically investigated.ResultsThe mesopancreas strains itself dorsally of the mesenteric vessels as a whitish-firm, fatty tissue-like layer. Macroscopically, in the dissected en-bloc specimens of pancreas and mesopancreas nerve plexuses were found running from the dorsal site of the pancreatic head to the mesopancreas to establish a perineural plane. Immunohistochemical examinations revealed the lymphatic vessels localized in direct vicinity of the neuronal plexuses between pancreas and mesopancreas.ConclusionThe mesopancreas as a perineural lymphatic layer located dorsally to the pancreas and reaching beyond the mesenteric vessels has not been classified in the anatomical or surgical literature before. The aim to ensure the greatest possible distance from the retropancreatic lymphatic tissue which drains the carcinomatous focus can be achieved in patients with pancreatic cancer only by complete resection of the mesopancreas (RMP).

Submicron Scale-Structured Hydrophilic Titanium Surfaces Promote Early Osteogenic Gene Response for Cell Adhesion and Cell Differentiation

BACKGROUND AND PURPOSE Titanium (Ti) surface roughness and surface hydrophilicity are key factors to regulate osteogenic cell responses during dental implant healing. In detail, specific integrin-mediated interactions with the extracellular environment trigger relevant osteogenic cell responses like differentiation and matrix synthesis via transcriptions factors. Aim of this study was to monitor surface-dependent osteogenic cell adhesion dynamics, proliferation, and specific osteogenic cell differentiation over a period of 7 days. MATERIALS AND METHODS Ti disks were manufactured to present smooth pretreatment (PT) surfaces and rough sandblasted/acid-etched (SLA) surfaces. Further processing to isolate the uncontaminated TiO(2) surface from contact with atmosphere provided a highly hydrophilic surface without alteration of the surface topography (modSLA). Tissue culture polystyrene (TCPS) served as control. Human osteogenic cells were cultivated on the respective substrates. After 24 hours, 48 hours, 72 hours, and 7 days, cell morphology on the Ti substrates was visualized by scanning transmission electron microscopy. As a marker of cellular proliferation, cell count was assessed. For the analysis of cell adhesion and differentiation, specific gene expression levels of the integrin subunits β1 and αv, runx-2, collagen type Iα (COL), alkaline phosphatase (AP), and osteocalcin (OC) were obtained by real-time RT-PCR for the respective time points. Data were normalized to internal controls. RESULTS TCPS and PT surfaces preserved a rather immature, dividing osteogenic phenotype (high proliferation rates, low integrin levels, and low specific osteogenic cell differentiation). SLA and especially modSLA surfaces promoted both cell adhesion as well as the maturation of osteogenic precursors into post-mitotic osteoblasts. In detail, during the first 48 hours, modSLA resulted in lowest cell proliferation rates but exhibited highest levels of the investigated integrins, runx-2, COL, AP, and OC. CONCLUSION Our results revealed a strong synergistic effect between submicron-scale roughness and surface hydrophilicity on early osteogenic cell adhesion and maturation.

Forces and deformations of the abdominal wall--a mechanical and geometrical approach to the linea alba.

Force-elongation responses of the human abdominal wall in the linea alba region were determined by tensile tests in which the linea alba was seen to exhibit a nonlinear elastic, anisotropic behavior as is frequently observed in soft biological tissues. In addition, the geometry of the abdominal wall was determined, based on MRI data. The geometry can be specified by principal radii of curvature in longitudinal of approximately 470 mm and in the transverse direction of about 200 mm. The determined radii agree with values found in other studies. Mechanical stresses, deformations and abdominal pressures for load cases above 6% elongation can be related using Laplaces formula and our constitutive and geometrical findings. Results from uni- and biaxial tensile tests can thus be compared using this model. Calculations confirm that abdominal pressures of approximately 20 kPa correspond to related biaxial forces of about 3.4N/mm in the transverse and 1.5 N/mm in the longitudinal direction. Youngs moduli can be calculated with respect to the uniaxial as well as the biaxial loading. At these physiological loadings, a compliance ratio of about 2:1 between the longitudinal and transversal directions is found. Youngs moduli of about 50 kPa occur in transversal direction and of about 20 kPa in longitudinal direction at transverse and longitudinal strains both in the order of 6%. These findings coincide with results from other investigations in which the properties of the abdominal wall have been examined.

How to Optimize Autonomic Nerve Preservation in Total Mesorectal Excision: Clinical Topography and Morphology of Pelvic Nerves and Fasciae

BackgroundUrogenital dysfunction after rectal and pelvic surgery was significantly decreased with the introduction of nerve-preserving dissection and total mesorectal excision (TME). Profound topographic knowledge of the pelvic connective tissue spaces is indispensable for identification and preservation of autonomic pelvic nerves. The purpose of this cadaver study was to highlight the course of important autonomous nerve structures and to identify potential injury sites.MethodsEleven cadavers were dissected according to TME with subsequent preparation of the pelvic nerves. The pelves of further three cadavers were sliced horizontally and cubed. Specimens were harvested and processed for light microscopy and immunohistochemistry to analyze both fascia and the types of nerves and their localization.ResultsThe neurovascular bundle, arising from the inferior pelvic plexus, shows the highest nerve density. At the lateral edge of Denonvilliers’ fascia, it pierces the parietal pelvic fascia. Several fine nerve branches spread into the loose periprostatic tissue up to the prostate or pass the prostate toward the urinary bladder. En route, we consistently find perikarya of autonomic nerves. Within the mesorectum, nerve fibers are distributed heterogeneously with laterally high densities, ventrally and dorsally low densities.ConclusionThe highest risk for pelvic nerve damage—apart from lesions of the superior hypogastric plexus itself—is anterolaterally of the rectum where the neurovascular bundle releases from the pelvic sidewall. Careful dissection helps to identify and protect these nerve structures. The retroprostatic Denonvilliers’ fascia contains no important nerve structures.

Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a−/− and Mep1b−/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.