Regression and persistence: remodelling in a tissue engineered axial vascular assembly

Abstract

In later stages of vasculoangiogenesis a vascular network is going through a metamorphosis for optimal perfusion and economy of energy. In this study we make a quantitative approach to phenomena of remodelling in a bioartificial neovascular network and suggest variance of calibre as a parameter of neovascular maturation. For this study, 18 male Lewis rats were subjected to the AV loop operation in combination with a hard porous biogenic matrix and an isolation chamber. The animals were allocated into three groups for different explantation intervals set to 2, 4 and 8 weeks, respectively. Collective attributes like vascular density, percent fractional area and variance of calibre were evaluated for a predefined region of interest (ROI). Late morphogenesis was evaluated by means of scanning electron microscopy. After the fourth week the absolute number of vessels within the ROI decreased (P < 0.03) whereas, on the contrary, the fractional area of all segments increased (P < 0.02). The variance in calibre was significantly increased in the 8-week group (P < 0.05). Lymphatic growth after week 4, early pericyte migration as well as intussusceptive angiogenesis were identified immunohistologically. Phenomena of remodelling were evaluated quantitatively in a neovascular network and variance could be proposed as a parameter of net vascular maturation.

Figures

Fig 1

(A) For construction of the AV loop, a segment of the right femoral vein is being harvested and interposed between the left femoral artery and vein. (B) The AV loop is placed in the peripheral groove of the porous matrix. Neovascular sprouts enter the matrix and render it vascularized. The construct is placed into an isolation chamber with an aperture for entrance of the vascular pedicle of the AV loop. (C) Method of analysis: A ROI was defined in a 500 μm radius (r) around the vascular axis (A), excluding the loop vessel itself. The image was rendered bimodal with a standardized threshold set to black stained India ink-filled vessels. The bimodal images were detected and analyzed for % FA of vessels against ROI (% FA), absolute capillary number (CapNo) in ROI as well as differential vascular calibre in ROI. In this way a developing vascular segment rather than an expanding one was evaluated. (D) Example on an acquired image.

Fig 2

(A) Venous segment, 2 weeks: The vascular network consists of numerous but small vessels, characteristic for an expanding neovascular system. The vascular axis filled with India ink gel is seen in the middle (HE, ×100). (B) Venous segment, 8 weeks: The number of vessels is not significantly higher but the calibre varies greatly building a vascular hierarchy from arterioles and precapillary arterioles, to gas-exchanging capillaries, postcapillary venules and venules draining into veins (HE, ×100). (C) Arterial segment, 4 weeks. Around the artery the neovascularization is sparse. The artery possesses a distinct tunica elastica and a thick muscular layer (van Gieson-Verhoeff, ×100). (D) Identical specimen as in 2c, venous segment. A marked difference from the arterial segment can be observed in terms of luminal calibre, variance and number. The vein shows some intimal hyperplasia concomitant with the arterialization process after construction of the AV loop. (van Gieson-Verhoeff, ×100). (E) Venous segment, 4 weeks. PCT stain red, endothelial cells and polymorphonuclear cells stain blue. PCT provide stabilization of the endothelium-their presence is thought to be a sign of maturation in a vessel (α-SM actin, ×250). (F) Lectin staining of endothelial cells demonstrates functionally full-grown vessels. On the upper right corner of the image there are three vascular conduits positive for lectin but void of India ink. Such a finding might be indicative of lympnatics present as soon as 4 weeks after initiation of neovascularization (lectin, ×350).

Fig 3

(A) and (B) Serial sections of a 2-week specimen. (A) In the middle of the image, a neovascular ‘tripode’ (arrow) with PCT (red staining) encircling the endothelial formation (blue staining of nuclei) shows no India ink filling. On the right side (B) the formation shows clearly endothelial staining positive for lectin, with still no India ink filling. This finding is indicative of a vessel furnished with PCT prior to formation of its lumen. Because lumen formation is a relatively early phenomenon of angiogenesis, it seems like pericytic migration and endothelial proliferation are parallel mechanisms, as postulated recently (see text). 4a: α-SM actin, ×350; 4b: lectin; serial sections. (C) and (D) Non-sprouting angiogenesis by means of intussusceptive microvascular growth (IMG) is an efficient modus for expansion of a neovascular network. In this process a vessel is divided into two vessels by ingrowth (intussusception) of an interstitial pillar. On the right side (D) this process is demonstrated on a microvascular replica under the scanning electron microscope. If these pillars are of endothelial or fibroblastic origin is not fully understood. Asterisk: Early stage; Arrow advanced stage showing formation of a new vascular conduit. On the left side (C) a venuole is interrupted by an endothelial bridge at the asterisk. One could assume that intussusception is a phenomenon carried out by the endothelial cells; however, remodelling of angiogenesis is grossly a three-dimensional process and is best demonstrated by three-dimensional methods. 3c: (α-SM actin, ×200); 3d: SEM of corrosion casts, ×230.

Fig 4

Left: Cross sectional area of vascular lumina in per cent (% FA) of total area of ROI detected in a radius 500 μm around the vascular axis. Development in all separate segments of the AV loop (vein, graft, artery) over the time intervals 2, 4 and 8 weeks. There is a steady increase over time in all segments indicative of a rise of the summation of the calibre and perfusion volume of all detected vessels. The highest per cent values are met with the vein, demonstrating the highest vasculoangiogenic activity in this region. After 8 weeks values for % FA are comparable all three segments, indicating achievement of a steady state. Right: Number of detected vascular lumina within the ROI. Past the 4th week, the number of lumina decreases in the non-arterial segments of the AV loop. Involution of a nascent vascular network in terms of regression of superfluous vessels happens in favour of necessary vascular conduits that persist to form feeding arterioles and draining venules, As angiogenesis in the vicinity of the artery is not as vivid as in the non arterial counterparts, phenomena of remodelling also take place later. For each bar n= 5 with 10 independent measurements, (*): P < 0.05. Error indicators show standard error of means.

Fig 5

Luminal diameters (calibre) of individual vessels within a ROI were measured and the variance of calibre was extracted for each ROI. In an attempt to evaluate variance of calibre as a morphologic indicator of maturation for a given neovascular network, this parameter was investigated for all time intervals. The 8-week group presented a significantly higher variance of vascular calibre than either of the 2- and 4-week groups. For each bar n= 5, 30 independent measurements, (*): P < 0.05. Error indicators show standard error of means.

Fig 6

(A) (SEM, 2 weeks) Capillary loop formation via IAR is accomplished by division of a nascent vascular conduit into two parallel ones. This process offers an energy saving alternative for optimalization of a vascular system. (B) (SEM, 2 weeks) Forward sprouting angiogenesis in a angiogenic ‘hot spot’. Asterisks represent neovascular sprouts. (C) (SEM, 8 weeks). The arterial or venous character of a vessel can be identified by the shape of the nuclear imprints on the cast. Long spindle-shaped endothelial nuclei are indicative for artery (A); rounded and planar nuclear imprints indicate a vein (V). Here, vessels demonstrate an arterial or venous character in the 8-week replica. (D) (SEM, 4 weeks) Formation of intercapillary loops and detail (E). New lumina are generated between capillaries to serve as interconnection, where none exist. (F) (SEM, 4 weeks). IBR is a means of optimizing the branching in terms of branching angle and interbranching distance. Interstitial pillars form along the long axis of the vessels prior to the branching point.