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.

Prevention of cement leakage into the hip joint by a standard cement plug during PFN-A cement augmentation: a technical note.

Medial penetration of the helical blade into the hip joint after fixation of trochanteric fractures using the proximal femur nail antirotation (PFN-A) is a potential failure mode. In low demand patients a blade exchange with cement augmentation may be an option if conversion to total hip arthroplasty is unfeasible to salvage the cut-through. This article describes a technique to avoid intraarticular cement leakage using a cement plug to close the defect in the femoral head caused by the cut-through.

An anatomical investigation of the cervicothoracic ganglion.

Anatomical variability within the autonomic nervous system has long been accepted. This study evaluated the anatomical variability of the cervicothoracic ganglion (CTG) according to its form and, in addition, provided precise measurements between the CTG and the anterior tubercle of the transverse process of the sixth cervical vertebra (C6TP), the first costovertebral articulation, and the vertebral artery. Forty-two adult cadavers were dissected, 22 male and 20 females. Five main forms of CTG were documented; spindle (31.9%), dumbbell (23.2%), truncated (21.7%), perforated (14.5%), and inverted-L (8.7%). The means for length, width, and thickness of the CTG were 18.5 mm, 8.2 mm, and 4.5 mm, respectively. The dimensions were found to be slightly larger in the males than females and on the left sides as compared to the right. The mean shortest distance between the CTGs and the vertebral artery was found to be 2.8 mm, whilst the mean shortest distances to C6TP was 25.7 mm and to the first costovertebral articulation was 1.7 mm. There is great variability in the morphology of the CTG with five common forms consistently seen. The relation to the vertebral artery may influence the form of the ganglion. Two previously undocumented forms are recorded; the truncated which describes the important juxtaposition of the CTG and the vertebral artery and the perforated which describes the piercing of the ganglion itself by the artery. The findings are considered to be of clinical importance to anesthetists, surgeons, neurosurgeons, and anatomists.

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.

A novel family of serine/threonine kinases participating in spermiogenesis.

The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of approximately 65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.

Parabronchial angioarchitecture in developing and adult chickens.

The avian lung has a highly sophisticated morphology with a complex vascular system. Extant data regarding avian pulmonary angioarchitecture are few and contradictory. We used corrosion casting techniques, light microscopy, as well as scanning and transmission electron microscopy to study the development, topography, and distribution of the parabronchial vasculature in the chicken lung. The arterial system was divisible into three hierarchical generations, all formed external to the parabronchial capillary meshwork. These included the interparabronchial arteries (A1) that ran parallel to the long axes of parabronchi and gave rise to orthogonal parabronchial arteries (A2) that formed arterioles (A3). The arterioles formed capillaries that participated in the formation of the parabronchial mantle. The venous system comprised six hierarchical generations originating from the luminal aspect of the parabronchi, where capillaries converged to form occasional tiny infundibular venules (V6) around infundibulae, or septal venules (V5) between conterminous atria. The confluence of the latter venules formed atrial veins (V4), which gave rise to intraparabronchial veins (V3) that traversed the capillary meshwork to join the interparabronchial veins (V1) directly or via parabronchial veins (V2). The primitive networks inaugurated through sprouting, migration, and fusion of vessels and the basic vascular pattern was already established by the 20th embryonic day, with the arterial system preceding the venous system. Segregation and remodeling of the fine vascular entities occurred through intussusceptive angiogenesis, a process that probably progressed well into the posthatch period. Apposition of endothelial cells to the attenuating epithelial cells of the air capillaries resulted in establishment of the thin blood-gas barrier. Fusion of blood capillaries proceeded through apposition of the anastomosing sprouts, with subsequent thinning of the abutting boundaries and ultimate communication of the lumens. Orthogonal reorientation of the blood capillaries at the air capillary level resulted in a cross-current system at the gas exchange interface.

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.

The Pararectus approach provides secure access to the deep circumflex iliac vessel for harvest of a large sized and vascularized segment of the iliac crest.

BACKGROUND: The feasibility of harvesting a vascularized iliac crest utilizing the Pararectus approach was assessed in cadavers and then this new technique was implemented in a clinical case. METHODS: Bilaterally in five cadavers the branches of both external iliac arteries were injected with colored silicone to assess their position to each other and to harvest a bone graft vascularized by the deep circumflex iliac artery (DCIA) through the Pararectus approach. This technique was implemented in a 68-years-old female patient, initially admitted to a level-I-trauma center after sustaining multiple injuries by falling from great height. For definitive treatment of a severely contaminated medially open (Gustilo-Anderson Type 3A) calcaneal luxation fracture (Sanders type IIIBC) in this patient a vascularized iliac crest autograft harvest by the Pararectus approach was used for reconstructive surgery. RESULTS: The DCIA and the deep inferior epigastric vessels (DIEV: vascularizing the rectus abdominis muscle and main pedicle of the inferiorly based rectus abdominis myocutaneous flap) are very close on the lateral and medial border of the external iliac artery, respectively. As a consequence, the retrograde dissection of the DIEV towards the DCIA through the Pararectus approach made the dissection of the vascularized iliac crest more amenable, preserving both the lateral femoral cutaneous and the genitofemoral nerves. Four months after the surgery the patient was able to fully weight-bear in orthopedic shoes. Radiographs and CT scans showed correct hind foot alignment and bony integration of the vascularized iliac crest graft into the residual calcaneal body. CONCLUSION: The Pararectus approach allowed for secure collection of large vascularized iliac grafts. The presented technique was successful as a salvage procedure in a clinical case with substantial bone loss after an open calcaneal fracture.

Percutaneous screw fixation of the iliosacral joint: A case-based preoperative planning approach reduces operating time and radiation exposure.

INTRODUCTION: A preoperative planning approach for percutaneous screw fixation of the iliosacral joint provides specific entry points (EPs) and aiming points (APs) of intraosseous screw pathways (as defined by CT scans) for lateral fluoroscopic projections used intraoperatively. The potential to achieve the recommended EPs and APs, to obtain an ideal screw position (perpendicular to the iliosacral joint), to avoid occurrence of extraosseous screw misplacement, to reduce the operating time and the radiation exposure by utilizing this planning approach have not been described yet. METHODS: On preoperative CT scans of eight human cadaveric specimen individual EPs and APs were identified and transferred to the lateral fluoroscopic projection using a coordinate system with the zero-point in the center of the posterior cortex of the S1 vertebral body (x-axis parallel to upper S1 endplate). Distances were expressed in relation to the anteroposterior distance of the S1 upper endplate (in%). In each specimen on one side a screw was placed with provided EP and AP (New Technique) whereas at the contralateral side a screw was placed without given EP and AP (Conventional Technique). Both techniques were compared using postoperative CT scans to assess distances between predefined EPs and APs and the actually obtained EPs and APs, screw angulations in relation to the iliosacral joint in coronal and axial planes and the occurrence of any extraosseous screw misplacement. The "operating time (OT)" and the "time under fluoroscopy (TUF)" were recorded. Statistical analysis was performed by the Wilcoxon signed-rank test. RESULTS: EPs were realized significantly more accurate using the new technique in vertical direction. The screw positions in relation to the iliosacral joint showed no significant difference between both techniques. Both techniques had one aberrantly placed screw outside the safe corridor. The (mean±SD) "OT" and the (mean±SD) "TUF" were significantly decreased using the new technique compared to the conventional technique (OT: 7.6±2min versus 13.1±5.8min, p=0.012; TUF: 1.5±0.8min versus 2.2±1.1min). CONCLUSION: The presented preoperative planning approach increases the accuracy in percutaneous screw fixation of the iliosacral joint, reduces operating time and minimizes radiation exposure to patient and staff.

Intussusceptive angiogenesis: its role in embryonic vascular network formation.

Intussusceptive angiogenesis is a novel mode of blood vessel formation and remodeling, which occurs by internal division of the preexisting capillary plexus without sprouting. In this study, the process is demonstrated in developing chicken eye vasculature and in the chorioallantoic membrane by methylmethacrylate (Mercox) casting, transmission electron microscopy, and in vivo observation. In a first step of intussusceptive angiogenesis, the capillary plexus expands by insertion of numerous transcapillary tissue pillars, ie, by intussusceptive microvascular growth. In a subsequent step, a vascular tree arises from the primitive capillary plexus as a result of intussusceptive pillar formation and pillar fusions, a process we termed "intussusceptive arborization." On the basis of the morphological observations, a 4-step model for intussusceptive arborization is proposed, as follows: phase I, numerous circular pillars are formed in rows, thus demarcating future vessels; phase II, formation of narrow tissue septa by pillar reshaping and pillar fusions; phase III, delineation, segregation, growth, and extraction of the new vascular entity by merging of septa; and phase IV, formation of new branching generations by successively repeating the process, complemented by growth and maturation of all components. In contrast to sprouting, intussusceptive angiogenesis does not require intense local endothelial cell proliferation; it is implemented primarily by rearrangement and attenuation of the endothelial cell plates. In summary, transcapillary pillar formation, ie, intussusception, is a central and probably widespread process, which plays a role not only in capillary network growth and expansion (intussusceptive microvascular growth), but also in vascular plexus remodeling and tree formation (intussusceptive arborization).

Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer.

Hypoxia has long been recognized as detrimental to the successful treatment of malignant tumors with ionizing radiation. Because hypoxia-inducible factor (HIF)-1alpha plays an essential role in oxygen homeostasis in vitro, we explored the predictive potential of this factor in a cohort of 98 patients with squamous cell cancer of the oropharynx, who were treated by curative radiation therapy. Ninety-four % of the primary tumors showed overexpression of HIF-1alpha, relative to the surrounding tissue, as determined by immunohistochemistry. The degree of HIF-1alpha immunoreactivity correlated inversely with both the rate of complete remission of the primary tumor (odds ratio, 0.33; P = 0.03) and lymph node metastases (odds ratio, 0.34; P = 0.02) as well as with local failure-free survival (risk ratio, 2.15; P = 0.006), disease-free survival (risk ratio, 2.01; P = 0.008), and overall survival (risk ratio, 2.17; P = 0.002). The multivariate analysis revealed the predictive power of HIF-1alpha to be independent of other covariables. We conclude that HIF-1alpha is overexpressed in the vast majority of patients with squamous cell cancer of the oropharynx and that the degree of expression has predictive and prognostic significance in individuals undergoing curative radiation therapy.

Surgical exposures and options for instrumentation in acetabular fracture fixation: Pararectus approach versus the modified Stoppa.

BACKGROUND: As an alternative to the modified Stoppa approach, the Pararectus approach is used clinically for treatment of acetabular fractures involving the anterior column. The current study assessed the surgical exposure and the options for instrumentation using both of these approaches. METHODS: Surgical dissections were conducted on five human cadavers (all male, mean age 88 years (82-97)) using the modified Stoppa and the Pararectus approach, with the same skin incision length (10cm). Distal boundaries of the exposed bony surfaces were marked using a chisel. After removal of all soft-tissues, distances from the boundaries in the false and true pelvis were measured with reference to the pelvic brim. The exposed bone was coloured and calibrated digital images of each inner hemipelvis were taken. The amount of exposed surface using both approaches was assessed and represented as a percentage of the total bony surface of each hemipelvis. For instrumentation, a suprapectineal quadrilateral buttress plate was used. Screw lengths were documented, and three-dimensional CT reconstructions were performed to assess screw trajectories qualitatively. Wilcoxon's signed rank test for paired groups was used (level of significance: p<0.05). RESULTS: After utilization of the Pararectus approach, the distances from the farthest boundaries of exposed bone towards the pelvic brim were significantly higher in the false but not the true pelvis, compared to the modified Stoppa approach. The percentage (mean±SD) of exposed bone accessible after utilizing the Pararectus approach was 42±8%, compared to 29±6% using the modified Stoppa (p=0.011). In cadavers exposed by the Pararectus approach, screws placed for posterior fixation and as a posterior column screw were longer by factor 1.8 and 2.1, respectively (p<0.05), and screws could be placed more posteromedial towards the posterior inferior iliac spine or in line with the posterior column directed towards the ischial tuberosity. CONCLUSION: Compared to the modified Stoppa, the Pararectus approach facilitates a greater surgical access in the false pelvis, provides versatility for fracture fixation in the posterior pelvic ring and allows for the option to extend the approach without a new incision.

Expression and activity of cell cycle regulators during proliferation and programmed cell death in the mammary gland.

In the mammary gland distinct phases of proliferation, differentiation and programmed cell death of epithelial cells occur at defined stages of development. Here we show that the expression and activity of cell cycle regulators during normal and preneoplastic proliferation and programmed cell death are remarkably similar. In all cases we found elevated levels of a protein kinase A activity and of transcription factor AP-1, cFos and JunD being the major components of the AP-1 DNA binding complex. A correlation between cFos and JunD expression and chromosomal DNA fragmentation during programmed cell death was observed. Several genes associated with G1, including cyclin D1, D2 and D3 and c-fos, c-jun, junB, JunD, c-myc and p53, are induced in proliferating and in apoptotic mouse mammary tissue. Whereas the expression of these genes correlated with active proliferation of epithelial cells in terminal end buds during puberty, very little proliferation or DNA synthesis, but, instead, extensive apoptosis of epithelial cells, was observed during involution. Our results suggest that a G1-like state is associated with programmed cell death of mammary epithelial cells in vivo and that apoptosis occurs without S-phase induction.

Recombinant human erythropoietin induces intussusceptive microvascular growth in vivo.

The role of erythropoietin (Epo) in angiogenesis has not been completely clarified. Epo induces endothelial cell proliferation and migration and stimulates angiogenesis on rat aortic rings in vitro and in vivo in the chick embryo chorioallantoic membrane (CAM) assay. The aim of the present study was to evaluate the ultrastructural aspects of angiogenesis in the CAM vasculature after recombinant human Epo (rHuEpo) exposure. The results demonstrated that after rHuEpo stimulation, the generation of new blood vessels occurred more frequently following an intussusceptive microvascular growth (IMG) mechanism. We have performed our experiments between days 8 and 12 of incubation, that is, when in the normal condition the capillary network expands mainly by IMG, and because it is generally accepted that implants made from days 8 to 10 are strongly angiogenic. This response is peculiar of rHuEpo, because it is abolished when an Epo-blocking antibody was coadministered with Epo.

Genetic, Genomic and Epigenomic Studies of Balkan Endemic Nephropathy (Ben).

BEN is a primary, chronic tubulointerstitial nephritis characterized with chronic anemia, absence of edema, xantoderma, normal blood pressure and normal findings on the fundus oculi. The disease is distributed in restricted areas in Bulgaria, Romania, Croatia, Bosnia, Former Yugoslavia. Despite numerous studies on genetic and environmental factors and their possible involvement in BEN, its etiopathogenesis still remains elusive. Our recent study aim to elucidate the possible epigenetic component in BEN development. Whole genome DNA array methylation analysis was applied to compare the methylation profiles of male and female BEN patients from endemic regions in Bulgaria and Serbia and healthy controls. All three most prominent candidate genes with aberrations in the epigenetic profile discovered with this study are involved in the inflammatory/immune processes and oncogenesis. These data are in concordance with the reported pathological alterations in BEN. This research supports the role of epigenetic changes in BEN pathology. Exome sequencing of 22.000 genes with Illumina Nextera Exome Enrichment Kit revealed three mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients which encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

Intratumoral microvessel density predicts local treatment failure of radically irradiated squamous cell cancer of the oropharynx.

PURPOSE: To determine the predictive value of intratumoral microvessel density (IMD), and of the expression of p53, vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) for the radiocurability of patients with squamous cell cancer of the oropharynx. MATERIALS AND METHODS: 139 patients with squamous cell cancer of the oropharynx were radically irradiated (median dose, 74 Gy) between 1991 and 1997. Biopsies from 100 patients were processed for immunohistochemistry. IMD was determined in hot spot areas of tissue stained with anti-CD31 at a magnification of x200. Staining for p53 was considered positive if more than 10% of the cell nuclei overexpressed the protein. Immunostaining of VEGF and TSP-1 was assessed semiquantitatively. RESULTS: Increasing IMD (range, 54-282) was strongly correlated with incomplete remission of both the primary tumors (p = 0.01) and lymph node metastases (p = 0.02). Moreover, multivariate Cox regression analysis revealed local failure-free survival to decline with increasing IMD (IMD continuous: risk ratio = 1.01 per increase of 1 microvessel, p = 0. 0001; IMD categorical: 130: risk ratio = 13.01). Neither the expression of p53, nor that of VEGF or TSP-1 was associated with the treatment outcome or IMD, but VEGF and TSP-1 expression were positively correlated (p = 0.02). CONCLUSION: IMD represents a powerful and independent predictive factor for local treatment failure in radically irradiated patients with squamous cell cancer of the oropharynx.

TGF-beta 3 expression correlates with epithelial cell death in normal, hyperplastic and malignant prostate.

Cytokines of the TGF beta family are thought to be involved in cellular growth control and are therefore likely candidates to regulate homeostasis of the prostate. We have analyzed immunohistochemically the expression of TGF-beta 3 in normal prostate (NP), benign prostate hyperplasia (BPH) and prostate cancer (PCa). Its expression was correlated to cell death and cell proliferation using double labeling techniques with terminal transferase and anti-Ki67 antibodies, respectively. TGF-beta 3 expression, localized to the basal cell layer of glandular epithelium, was found in NP and BPH. In TGF-beta 3 positive regions cell death was frequently detected, while proliferating cells were only observed in TGF-beta 3 negative areas. Moreover, cell death was not observed in the absence of TGF-beta 3. PCa was characterized by high cell proliferation and the absence of cell death. TGF-beta 3 expression could not be detected in PCa. Hormonal ablation is the main therapeutic protocol used today suffering, however, from a high relapse rate. We have used the rat as a model system to show that castration, resulting in massive cell death of glandular epithelial cells, induces overall expression of TGF-beta 3 in the basal cell layers. Interestingly, investigation of tumor material from patients received after hormonal ablation revealed the simultaneous presence of TGF-beta 3 positive, hyperplastic regions undergoing cell death and TGF-beta 3 negative highly proliferating malignant foci. Our results suggest that the expression of TGF-beta 3 strictly correlates with cell death in normal and hyperplastic prostate and that disappearance of TGF-beta 3 indicates high cell proliferation and the establishment of the malignant phenotype.

Recapitulation of a normal cellular growth program in early invasive breast-cancer.

With a view to identifying markers reflecting not the evidence of, but the potential for, neoplastic progression of the breast we have compared the normal invasive mammary epithelial cell growth seen at puberty with invasive and non-invasive carcinogenesis using the mouse as a model. We have analyzed cell proliferation, the expression of the metalloproteinase stromelysin-1 and of the extracellular matrix protein tenascin. Striking parallels were observed between pubertal growth and the development of invasive, metastasizing mouse mammary tumors. In particular, the myoepithelial to epithelial transition of proliferation and stromelysin-1 expression was a hallmark of both normal growth at puberty and the early development of aggressive tumors. Investigation of neoplastic lesions in the human breast indicated that the pubertal growth characteristics are recapitulated only in the development of ductal carcinomas and may define early stages of invasive and potentially malignant growth.

Vascular remodelling during the normal and malignant life cycle of the mammary gland.

The mammary gland life cycle is exemplified by massive, physiologically dictated changes in cell number and composition, architecture, and functionality. These drastic upheavals, by necessity, also involve the mammary endothelium, which undergoes angiogenic expansion during pregnancy and lactation followed by ordered regression during involution. In this review, we summarise data obtained using the Mercox methyl methacrylate corrosion cast technique to analyse the mammary gland vasculature during normal development and carcinogenesis. Concomitant with epithelial cell expansion, the mammary vasculature grows during the first half of pregnancy by sprouting angiogenesis whereas the last half of pregnancy and lactation are characterised by the non-proliferative intussusceptive angiogenesis. The vasculature of the lactating gland is composed of a well-developed capillary meshwork enveloping the secretory alveoli with basket-like honeycomb structures. During involution, regression of the vasculature is achieved by regional collapse of the honeycomb structures, capillary retraction, and endothelial attenuation. This process appears partly to involve apoptosis. However, an additional mechanism involving remodelling without cell death, which we have termed angiomeiosis, must exist to explain the morphological observations. Interestingly, in mammary tumours of neuT transgenic mice, both sprouting and intussusceptive angiogenesis was observed simultaneously in the same nodules, a finding with potential implications for cancer therapy. The underlying molecular mechanisms controlling angiogenic modulation in the mammary gland, particularly angiogenic regression and the endothelial:parenchymal interplay, are poorly understood. However, the data summarised in this review indicate that precisely these molecular mechanisms offer novel alternatives for specific and effective treatment of breast cancer.

Intussusceptive arborization contributes to vascular tree formation in the chick chorio-allantoic membrane.

Various reports indicate that the process of intussusceptive microvascular growth (IMG) plays a crucial role in capillary network formation of the chorio-allantoic membrane (CAM). In the present study we demonstrate by methylmethacrylate (Mercox) casting and in vivo time-lapse observations that intussusception, i.e. insertion of transcapillary tissue pillars, is also strongly involved in vascular tree formation, a process we refer to as intussusceptive arborization (IAR). From day 7 to day 14 of incubation, several arterial and venous branching generations arise from the capillary plexus. The process is initiated by pillar formation in rows, which are demarcating future large vessels in the capillary meshwork. In a subsequent step the pillars undergo reshaping to form narrow tissue septa that successively merge, which results in the production of new generations of blood vessels. This is followed by growth and maturation of all vascular components. The process of IAR in the CAM is very active at days 10 and 11 of incubation and takes place in preferentially perfused capillary regions determining "dynamic areas". The process of intussusception may be preceded by endothelial division, but the transcapillary pillar formation itself occurs primarily by rearrangement and attenuation of the endothelial cells without local endothelial cell proliferation. We conclude that after the early sprouting phase, the process of intussusception is the basic mechanism of CAM vascularization. It leads to capillary network growth and expansion (IMG) and, at the same time to feed vessel formation with several branching generations (IAR).