Andrew N. Makanya

Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling

New blood vessels arise initially as blood islands in the process known as vasculogenesis or as new capillary segments produced through angiogenesis. Angiogenesis itself encompasses two broad processes, namely sprouting (SA) and intussusceptive (IA) angiogenesis. Primordial capillary plexuses expand through both SA and IA, but subsequent growth and remodeling are achieved through IA. The latter process proceeds through transluminal tissue pillar formation and subsequent vascular splitting, and the direction taken by the pillars delineates IA into overt phases, namely: intussusceptive microvascular growth, intussusceptive arborization, and intussusceptive branching remodeling. Intussusceptive microvascular growth circumscribes the process of initiation of pillar formation and their subsequent expansion with the result that the capillary surface area is greatly enhanced. In contrast, intussusceptive arborization entails formation of serried pillars that remodel the disorganized vascular meshwork into the typical tree-like arrangement. Optimization of local vascular branching geometry occurs through intussusceptive branching remodeling so that the vasculature is remodeled to meet the local demand. In addition, IA is important in creation of the local organ-specific angioarchitecture. While hemodynamic forces have proven direct effects on IA, with increase in blood flow resulting in initiation of pillars, the preponderant mechanisms are unclear. Molecular control of IA has so far not been unequivocally elucidated but interplay among several factors is probably involved. Future investigations are strongly encouraged to focus on interactions among angiogenic growth factors, angiopoetins, and related receptors.

Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after cessation of treatment. In numerous preclinical studies, angiogenesis inhibitors were shown to be efficient in the treatment of many pathological conditions, including solid cancers. In most clinical trials, however, this approach turned out to have no significant effect, especially if applied as monotherapy. Recovery of tumors after therapy is a major problem in the management of cancer patients. The mechanisms underlying tumor recovery (or therapy resistance) have not yet been explicitly elucidated. This review deals with the transient switch from sprouting to intussusceptive angiogenesis, which may be an adaptive response of tumor vasculature to cancer therapy that allows the vasculature to maintain its functional properties. Potential candidates for molecular targeting of this angioadaptive mechanism are yet to be elucidated in order to improve the currently poor efficacy of contemporary antiangiogenic therapies.

New insights into intussusceptive angiogenesis.

Angiogenesis is defined as the growth and development of new capillary blood vessels from pre-existing vasculature [1]. It is requisite in metazoans because the transfer of nutrients and wastes has to be accomplished by diffusion through the tissue and hence, the respiring cells need to be within 100–200 μm of the blood vessels, which is the diffusion limit for oxygen [2]. Angiogenesis is a normal process fundamental in wound healing, reproduction and development. Abnormal angiogenic activity occurs in non-neoplastic diseases such as arthritis, psoriasis, trachoma, and diabetic retinopathy and in the vascularisation of tumours (for details, see [3–8]). Angiogenesis has two facets: sprouting angiogenesis and intussusceptive angiogenesis. This chapter concentrates on the basic mechanisms, facets and outcomes of intussusceptive angiogenesis.

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.

The structural design of the bat wing web and its possible role in gas exchange

The structure of the skin in the epauletted fruit bat (Epomophorus wahlbergi) wing and body trunk was studied with a view to understanding possible adaptations for gas metabolism and thermoregulation. In addition, gas exchange measurements were performed using a respirometer designed for the purpose. The body skin had an epidermis, a dermis with hair follicles and sweat glands and a fat‐laden hypodermis. In contrast, the wing web skin was made up of a thin bilayered epidermis separated by a connective tissue core with collagen and elastic fibres and was devoid of hair follicles and sweat glands. The wings spanned 18–24 cm each, with about 753 cm2 of surface exposed to air. The body skin epidermis was thick (61 ± 3 µm, SEM), the stratum corneum alone taking a third of it (21 ± 3 µm). In contrast, the wing web skin epidermis was thinner at 9.8 ± 0.7 µm, with a stratum corneum measuring 4.1 ± 0.3 µm (41%). The wing capillaries in the wing web skin ran in the middle of the connective tissue core, with a resultant surface‐capillary diffusion distance of 26.8 ± 3.2 µm. The rate of oxygen consumption (V̇O2) of the wings alone and of the whole animal measured under light anaesthesia at ambient temperatures of 24 ºC and 33 ºC, averaged 6% and 10% of the total, respectively. Rate of carbon dioxide production had similar values. The membrane diffusing capacity for the wing web was estimated to be 0.019 ml O2 min−1 mmHg−1. We conclude that in Epomophorus wahlbergi, the wing web has structural modifications that permit a substantial contribution to the total gas exchange.

Morphological analysis of the postnatally developing marsupial lung: The quokka wallaby

We investigated the events that take place during the postnatal morphogenesis of the lung of the quokka wallaby, Setonix brachyurus, using the light microscope and both the scanning and transmission electron microscopes. The lung of term, newborn babies (joeys) at 3‐days of postnatal life was at late canalicular stage and comprised large airways and tubules separated by thick mesenchymal interstitium. The tubules were lined by a low cuboidal epithelium but had few portions with true gas exchange barrier where capillaries came into close contact with squamous type of epithelium. By the fifth day postpartum, the lung entered the early saccular stage characterised by large air sacs, thinner septa, a better developed double capillary system and conversion of the cuboidal epithelium into a squamous one of type I cells interrupted by groups of cuboidal type II cells with lamellar bodies. Transitory respiratory bronchioles were recognisable toward the end of this stage. Formation of secondary septa started by Day 15, dividing the saccules into several generations of smaller air spaces. There were alternating and concurrent periods of tissue proliferation and air space expansion, followed by septal thinning. Alveolization started from about 125 days postpartum when the first burst of small sized air spaces bounded by septa with a single capillary layer were encountered. By Day 180 the process of alveolization was completed with only occasional septa showing a double capillary system and by Day 210 postnatally, the lung resembled that of an adult. For the first time in a mammal, the canalicular stage was encountered postnatally during lung development. Anat Rec 262:253–265, 2001.

Decrease in VEGF Expression Induces Intussusceptive Vascular Pruning

Objective—The concept of vascular pruning, the “cuting-off” of vessels, is gaining importance due to expansion of angio-modulating therapies. The proangiogenic effects of vascular endothelial growth factor (VEGF) are broadly described, but the mechanisms of structural alterations by its downregulation are not known. Methods and Results—VEGF165-releasing hydrogels were applied onto the chick chorioallantoic membrane on embryonic day 10. The hydrogels, designed to completely degrade within 2 days, caused high-level VEGF presentation followed by abrupt VEGF withdrawal. Application of VEGF resulted in a pronounced angiogenic response within 24 hours. The drastic decrease in level of exogenous VEGF-A within 48 hours was corroborated by enzyme-linked immunosorbent assay. Following this VEGF withdrawal we observed vasculature adaptation by means of intussusception, including intussusceptive vascular pruning. As revealed on vascular casts and serial semithin sections, intussusceptive vascular pruning occurred by emergence of multiple eccentric pillars at bifurcations. Time-lapse in vivo microscopy has confirmed the de novo occurrence of transluminal pillars and their capability to induce pruning. Quantitative evaluation corroborated an extensive activation of intussusception associated with VEGF withdrawal. Conclusion—Diminution of VEGF level induces vascular tree regression by intussusceptive vascular pruning. This observation may allude to the mechanism underlying the “normalization” of tumor vasculature if treated with antiangiogenic drugs. The mechanism described here gives new insights into the understanding of the processes of vasculature regression and hence provides new and potentially viable targets for antiangiogenic and/or angio-modulating therapies during various pathological processes.

Comparative functional structure of the olfactory mucosa in the domestic dog and sheep

Olfactory acuity differs among animal species depending on age and dependence on smell. However, the attendant functional anatomy has not been elucidated. We sought to determine the functional structure of the olfactory mucosa in suckling and adult dog and sheep. Mucosal samples harvested from ethmoturbinates were analyzed qualitatively and quantitatively. In both species, the olfactory mucosa comprised olfactory, supporting and basal cells, and a lamina propria containing bundles of olfactory cell axons, Bowmans glands and vascular elements. The olfactory cells terminated apically with an expanded knob, from which cilia projected in a radial fashion from its base and in form of a tuft from its apex in the dog and the sheep respectively. Olfactory cilia per knob were more numerous in the dog (19 ± 3) compared to the sheep (7 ± 2) (p<0.05). In the dog, axonal bundles exhibited one to two centrally located capillaries and the bundles were of greater diameters (73.3 ± 10.3 μm) than those of the sheep (50.6 ± 6.8 μm), which had no capillaries. From suckling to adulthood in the dog, the packing density of the olfactory and supporting cells increased by 22.5% and 12.6% respectively. Surprisingly in the sheep, the density of the olfactory cells decreased by 26.2% while that of the supportive cells showed no change. Overall epithelial thickness reached 72.5 ± 2.9 μm in the dog and 56.8 ± 3.1 μm in the sheep. These observations suggest that the mucosa is better structurally refined during maturation in the dog than in the sheep.

Development and spatial organization of the air conduits in the lung of the domestic fowl, Gallus gallus variant domesticus.

We employed macroscopic and ultrastructural techniques as well as intratracheal casting methods to investigate the pattern of development, categories, and arrangement of the air conduits in the chicken lung. The secondary bronchi included four medioventral (MVSB), 7–10 laterodorsal (LDSB), 1–3 lateroventral (LVSB), several sacobronchi, and 20–60 posterior secondary bronchi (POSB). The latter category has not been described before and is best discerned from the internal aspect of the mesobronchus. The secondary bronchi emerged directly from the mesobronchus, except for the sacobronchi, which sprouted from the air sacs. Parabronchi from the first MVSB coursed craniodorsally and inosculated their cognates from the first two LDSB. The parabronchi from the rest of the LDSB curved dorsomedially to join those from the rest of the MVSB at the dorsal border. Sprouting, migration, and anastomoses of the paleopulmonic parabronchi resulted in two groups of these air conduits; a cranial group oriented rostrocaudally and a dorsal group oriented dorsoventrally. The neopulmonic parabronchial network formed through profuse branching and anastomoses and occupied the ventrocaudal quarter of the lung. There were no differences in the number of secondary bronchi between the left and right lungs. Notably, a combination of several visualization techniques is requisite to adequately identify and enumerate all the categories of secondary bronchi present. The 3D arrangement of the air conduits ensures a sophisticated system, suitable for efficient gas exchange. Microsc. Res. Tech., 2008.

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 α‐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. Developmental Dynamics 235:68–81, 2006.