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(Circulation Research. 2001;89:723.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Vascular Morphogenesis and Remodeling in a Model of Tissue Repair

Blood Vessel Formation and Growth in the Ovarian Pedicle After Ovariectomy

Sybill Patan, Lance L. Munn, Shigeru Tanda, Sylvie Roberge, Rakesh K. Jain, Rosemary C. Jones

From the Departments of Radiation Oncology (S.P., L.L.M., S.T., S.R., R.K.J.) and Anesthesia (R.C.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Dr S. Patan, Department of Cardiology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Ave, Bronx, NY 10461. E-mail spatan,aecom.yu.edu

	Abstract

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Abstract—— To investigate mechanisms of vascular morphogenesis in tissue repair, we performed ovariectomy with resection of the corresponding branches of the ovarian vessels in nude mice. This induces a vascular network remodeling response in the healing ovarian pedicle. Reconstruction of 2000 histological serial sections demonstrated that a new vascular network composed of venous-venous loops forms in the wall of the dilated ovarian vein. Preexisting veins of all sizes, including a branch of the main artery, are subjected to segmentation. Loop formation and segmentation are based on intussusceptive microvascular growth. Loop formation is followed by elongation. Loop remodeling occurs also by intussusception and results in the formation of compound loop systems. All loop systems observed were completely patent. Blind-ending sprouts were extremely rare. Anastomoses between the preexisting vessels subjected to segmentation and the loop systems were established to include the newly formed vessels into the preexisting vascular network. The formation of an increasing number of patent loop systems likely decreases hypoxia and subsequently arrests angiogenesis with transformation of the granulation tissue into a scar. Loop formation also occurred inside a large thrombus that occluded a part of the lumen of the main vein.

Key Words: angiogenesis • endothelial cell • intussusceptive microvascular growth • tissue repair • restenosis

	Introduction

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Intussusceptive microvascular growth (IMG) is a concept of vascular morphogenesis and remodeling that provides an alternative mechanism to angiogenesis by endothelial sprouting.^1–10 The latter requires migration and proliferation of endothelial cells, the formation of blind-ending tubes and their connection to establish perfusion.^11–13,20 In contrast, IMG refers to partitioning of the perfused vessel lumen by the insertion of interstitial (or intervascular) tissue structures (ITSs, diameterg2.5 µm) or tissue pillars (or posts, diameterh2.5 µm).^1,3,6 This is followed by growth of these structures resulting in network expansion.¹⁴ IMG is also an important mechanism of vascular network remodeling, defined as rearrangement of the number and/or position of vascular segments without significant network expansion.^7,8

IMG consists of several cellular mechanisms as demonstrated by in vivo microscopy and analysis of light and electron microscopic serial sections. ^6–10 IMG has been detected in development in different organs and species^3–5,9,15 and in the pathological state in myocardial infarctions and tumor vascularization^7,16,16a (also Patan et al, unpublished data). The analysis of mice deficient of the tyrosine kinase receptor system TIE1, TIE2, and the TIE2 ligand, angiopoietin-1, revealed that endothelial cells were unable to stretch and recruit periendothelial cells to the vessel wall.^9,17 Correspondingly, TIE2- and angiopoietin-1–deficient mice exhibit a pathological structure of tissue pillars and tissue folds, the landmarks of IMG along with a coarse and uniform pattern of their vascular network.^9,17 This is indicative of a function in the molecular regulation of IMG.⁹

The goal of the present investigation was to analyze mechanisms of blood vessel formation in the adult organism by reconstruction of tissue serial sections. We used the "tissue isolated ovary" as a model of vascular morphogenesis in repair. After ovariectomy, the ovarian pedicle is exposed to a noninflammatory wound healing response, corresponding to the healing process of surgical wounds. Vascular morphogenesis and remodeling occur, based on severe alteration of the previous organ-specific perfusion pattern, in a simplified vascular network composed of a single artery and its corresponding vein (and their branches that supply the connective tissue of the pedicle, while the branches to the ovary are resected). This model was originally used for tumor perfusion studies with additional tumor cell injection.¹⁸

We detected abundant ITSs of very different sizes, the signs of IMG, in the main vein and its branches. Our analysis and reconstruction of different stages of these structures based on serial sections allow us to propose a new mechanism of blood vessel growth and formation occurring in tissue repair. This is based on "in situ formation" and remodeling of vascular loops by IMG.

	Materials and Methods

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Ovariectomy
Ovariectomy in nude mice was performed as previously described.¹⁹ The right ovary served as a control. In this study, three pedicles obtained on day 7 after ovariectomy were analyzed in great detail. Approximately 3 mm (length) of the ovarian vein was studied in 2000 serial sections (see Figure 1). Additionally, 2 pedicles of days 3, 14, and 21 each were screened. The mice were bred in the animal facility of the Edwin L. Steele Laboratory (Massachusetts General Hospital). The animals were treated according to the National Institutes of Health guidelines.

View larger version (62K): [in this window] [in a new window]   Figure 1. Spatial relationship of suture, main vein, loops derived from the main vein, and area of segmentation of smaller veins in the periphery of the pedicle, based on section numbers (A). Overview of the wound site (B, section 1716). Area surrounding the suture containing fibrin, leukocytes, and veins, which exhibit multiple signs of segmentation (C, section 1724). Asterisk indicates main ovarian vein; arrow, suture. Bar=400 µm (B); bar=90 µm (C).

Perfusion Fixation
The mice were anesthetized as described, and the isolated ovarian pedicles were fixed by perfusion with 2% paraformaldehyde in PBS (pH 7.4) through the abdominal aorta after ligation of all branches except the ovarian arteries. Fixative was drained through the caudal vena cava. Although perfusion fixation was extensively and thoroughly performed and the tissue was well preserved, many vessel profiles in several pedicles still contained different amounts of blood cells, indicating that the perfusate eventually bypassed parts of the extremely dense network composed of tiny segments (see Discussion).

Tissue Preparation for Light Microscopy
After perfusion, the ovariectomized pedicles were removed and postfixed in the same fixative overnight and embedded in resin at 4°C (Historesin, Leica) or paraffin. Serial 2-µm-thick sections (resin) were numbered with even numbers to match distances and stained with toluidine blue. Paraffin serial sections (3 µm thick) were single- and double-labeled with rat anti-mouse PECAM (CD31, Pharmingen) and mouse PCNA antibody (Zymed) according to manufacturers’ protocols. The antigens were visualized using the ABC peroxidase technique (kits from Vector and Zymed). All sections were viewed with an Olympus BX 40 microscope. Arterial and venous branches were distinguished based on histological criteria.

Computer Reconstruction of Vascular Networks in Serial Sections
Photographs of resin serial sections were digitized using a Scanjet 4P color scanner (Hewlett Packard) and contrast was enhanced. With the use of image-processing software (Adobe Photoshop 3.0), the red channel of the RGB images was subtracted from the green channel, resulting in a grayscale image of sufficient contrast to separate vessels from other tissue. Images were then subjected to a median filter to reduce noise and printed on transparency sheets. Registration marks were obtained by lining up relevant features on adjacent slices. The coordinates of 5 fiduciary points per slice were recorded and input to the NIH Image 1.59 software to register the slices and then project the volume using its built-in 3-D projection routine.

	Results

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Time Course of Vascular Morphogenesis and Remodeling in the Ovarian Pedicle
Vascular morphogenesis and remodeling after ovariectomy begin around day 3. For a precise time course, see the online supplementary information, available in the data supplement at http://www.circresaha.org.

IMG by Loop Formation
The dilated ovarian vein and some of its large branches are the major source of vascular morphogenesis by loop formation in the center of the granulation tissue between the main vein and the suture (Figures 1A through 1C). Analysis of serial sections demonstrates varying stages of this mechanism. The typical "elementary loop" forms the simplest loop structure and is initially only a few micrometers long. It is composed of a connecting segment, which originates from the main vein and is continuous with a second connecting segment that rejoins the vein. The tissue of the venous wall between both segments forms the center of the loop and corresponds in structure to a small ITS (Figures 2 and 7B). The analysis and reconstruction of serial sections reveal that this ITS or pillar, which is part of a tissue fold, remains connected to the fold at its bottom and top and is separated in between (Figures 2B and 2D, loop 1; 2C and 2E, loop 2). Figures 2, 3, and 4 show increasingly complex loop systems. In Figure 2, loop 1 is a single elementary loop, while loop 2 is in the process of forming a double loop at the margin of the main vein. In loop 1, an ITS is connected to the lateral wall of the main vein in Figures 2A, 2B, and 2D, but it is free, surrounded by the lumen at its entire circumference in Figure 2C, the intermediate section. Consequently, the lumen forms a loop around the ITS as shown in Figure 2C.

View larger version (105K): [in this window] [in a new window]   Figure 2. A through H (serial sections 3166[x20], 3166[x100], 3168, 3174, 3178, 3186, 3192, and 3194, respectively), Interstitial tissue structures (ITSs) define an elementary loop (loop 1, ITS: length=6 µm; diameter=33x18 µm, A through E) and a forming double-loop system (loop 2, ITS: length=8 µm; diameter=15x12 µm, A through H) originating from the wall of the main vein. Profiles of the ovarian vein filled with erythrocytes, and its derived loops, are seen in cross section. Loop 2 forms a short double-loop system, since it is composed of three connecting segments as illustrated by the inset. Additionally, the double loop contains another ITS (6 µm in diameter). Sectioned vertically, it connects the central ITS to the lateral venous wall and consequently divides the lumen of the loop (E). Corresponding segments depicted in Figures 2 and 3 are labeled with the same signs (yellow dot and star). Bar=60 µm (A); 30 µm (C through E); and 10 µm (B and F through H).

The forming double loop (2) demonstrates that elementary loops are transformed to more complex loop systems by splitting of the central ITS. Loop 2 consists of one elementary loop that surrounds a central ITS, similar to loop 1 (Figure 2D). After its insertion at two sides of the lateral venous wall (Figures 2E and 2F), the upper part of the ITS is split centrally by a third connecting segment (Figures 2G and 2H). The loop thus constitutes a small double loop, since the first connecting segment of the loop rejoins its vessel of origin via two other connecting segments (inset in Figure 2 and Figures 2D and 2H). As is evident in higher-magnification images, all ITSs are lined by endothelial cells and consist of a core of extracellular matrix that is rich in fibrin and contains extensions of fibroblasts (Figure 2B).

Elementary loops as shown in Figure 2 can elongate. Such a longer elementary loop (loop 3) that arises from the ovarian vein is illustrated in Figure 3. One connecting segment (a) projects from the ovarian vein into the surrounding tissue (Figure 3A) and separates from its mother vessel (Figure 3B). In consecutive sections, (a) projects toward the ovarian vein and reconnects to it through another connecting segment (Figures 3C through 3F). Loops 1 and 2 have common segments with loop 3. The three loops therefore share "common" connecting segments and form a small compound loop system (Figure 3H).

View larger version (100K): [in this window] [in a new window]   Figure 3. A through H (serial sections 3180, 3184, 3188, 3190, 3200, 3198, and 3206, respectively), Profiles of an elongated elementary loop (3) that is connected to loops 1 and 2 (Figure 2) are illustrated. Loops 1, 2 (Figure 2), and (3) share two common connecting segments: the first segment of loop (3) is identical with the left segment of loop 1 (asterisk in Figures 2 and 3); see also Figures 2C, 2E, and 3A. The second segment of (a) corresponds to the third segment of loop 2 (dot in Figures 2 and 3); see also Figures 2F through 2H and 3F. The ITS of loop 2 is depicted after its reinsertion at the lateral vessel wall where it is divided into two parts by the third loop segment (H). Bar=30 µm.

Further remodeling and elongation of these relatively small compound loop systems obviously lead to formation of large and complex ones. They are composed of one connecting segment that originates from the main vein and rejoins it via at least two other connecting segments (Figures 2D through 2H). In these compound systems, loops can also be superimposed onto each other (Figure 3H). A part of such a complex system of loops of varying lengths that originate from the ovarian vein, or from small loops derived from the latter, is illustrated in Figure 4 (serial sections 3074 through 3520). This system is documented by a sequence of photomicrographs of selected serial sections (Figures 4A through 4U), a graph (Figure 5), and a computer reconstruction of consecutive serial sections (online Figure 2). One vessel (b) forms five long loops in conjunction with its neighbor vessel (c) (Figure 5A: b-b₁-c₁-c, b-b₁-c₁-c₂-c, b-b₁-c₁-c₂-c, b-b₂-c₂-c, and b-b₃-b_1a-c₁-c₂-c). Additionally, it forms four short loops with its own branches (Figure 6: b₂-b₃-b₂, b₂-b_1a-b₁, and b₁-b_2a-b₂, b_2a-b₂). Only one blind-ending sprout (b₀) could be detected within this system (Figure 5). The neighboring vessel (c) also derives from the ovarian vein and represents a "self-looping" system. Thus its two branches (c₁) and (c₂) form 3 loops with each other. (c) also contains three elementary loops (segment c₁ in Figures 4F,4G, and 5). Finally, after several connections and separations of (b) and (c), vessel (c) joins (b). The main branches (b₁ and b₂) of vessel (b) then enter a part of the wound that is not yet as well organized compared with the previous one (Figure 4U). The other part of the compound loop system derived from vessels (b) and (c) is not illustrated. In this part (serial sections 2940 through 3074), (b) and (c) connect to form two loops and (c) contains two elementary loops, one being very small (diameter of the ITS=3 µm). Finally (c) connects to (b), which joins another large vessel also derived from the main vein, consequently forming a third loop. Thus we detected only one sprout in a system composed of 16 loops and no sprout within its second part, composed of 5 loops. Within two other systems, composed of 9 and 12 loops each, we also could not detect any sprout-like structures.

View larger version (334K): [in this window] [in a new window]   Figure 4. A through U (serial sections 3102, 3126, 3128, 3134, 3146, 3154, 3160, 3166, 3172, 3188, 3194, 3208, 3214, 3222, 3234, 3246, 3258, 3276, 3294, 3306, and 3514, respectively), Compound loop system (b-c) that originates from the main vein is illustrated by the profiles of its connecting segments. (a) is a segment of loop (a) depicted in Figure 3. (d) and (e) are segments of two other compound loop systems. This loop system starts with the separation of (b) and (c) from the main vein (A). Its main branches, b1 and b2, end in an area of tissue containing many extravasated blood cells and the occluded lumen of a larger venous branch in the area of the wound suture (U, arrow). Bar=80 µm.

View larger version (79K): [in this window] [in a new window]   Figure 5. Architecture of the compound loop system illustrated in Figure 4 based on reconstruction of sequential serial sections (3075 through 3300) spanning a distance of 225 µm. The vessel lumen is shown in red, the tissue in blue. Outside this system are numerous other compound loop systems that also derive from the main vein (not illustrated).

View larger version (59K): [in this window] [in a new window]   Figure 6. A through H, Vessel segmentation (serial sections 3128, 3130, 3132, 3136, 3138, 3142, 3144, 3150, respectively). The inset illustrates the architecture of the connected system of folds and ITSs (also labeled with yellow arrows and arrowheads) within the lumen. Bar=13 µm. I, Computer reconstruction of photomicrographs demonstrates vessel segmentation as shown in panels A through H (consecutive serial sections 3126 through 3150). The vessel diameter is 39 µm. J, Vascular connections between the peripheral vessels that are subjected to segmentation and the newly formed loop systems (arrowheads) around the main vein. Bar=400 µm.

The early transformation of a part of the main vein into a network of loops reveals how loop formation is initiated and promoted within the niches between tissue folds along the lateral venous wall (online Figure 3).

IMG by Segmentation
Segmentation, another mechanism of IMG, was detected in the main vein distant to the suture in front of the region of loop formation and in smaller veins immediately around the wound suture (Figures 1A through 1C). The latter area corresponds to the one of loop formation and granulation tissue. It is thus infiltrated by macrophages (Figures 1C and 6A through 6J). Dilated venous vessels located between fat cells contain numerous slender endothelial folds, which project into the center of the vessel lumen from various locations of the lateral vessel wall (Figures 6A through 6H). These folds frequently connect to others to form walls splitting the lumen (Figure 6F and inset to Figure 6). This process divides the vessel lumen completely or incompletely into subunits that form new vascular segments (Figure 6I). Out of 40 vessels in this area, 15 showed these signs of transformation. Importantly, these preexisting vessels exhibit numerous connections to the newly formed loop systems around the main vein (Figure 6J). Some of the inserted intervascular walls also split to form tissue pillars (Figures 6B and 6C). The folds are lined by thin extensions of endothelial cells that encircle tiny fibrin deposits and collagen fibrils within their centers and/or intraluminal tips. Larger folds also contain extensions of periendothelial cells. In precursor stages, some of these folds are yet not completely lined by endothelial cells. They are still composed of fibrin cords that are initially deposited in the vessel lumen to which leukocytes attach and along which endothelial cells of the vessel wall migrate (online Figure 4A). Different stages of segmentation were also detected in a large branch of the ovarian artery (online Figures 4B through 4E).

IMG in a Large Thrombus Located in the Main Vein
See the online supplementary information for more detail.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Different Mechanisms of Vascular Morphogenesis in the Healing Ovarian Pedicle
The present study identifies IMG as a major mechanism of vascular morphogenesis and remodeling in the ovarian pedicle after ovariectomy. In this model system, vascular morphogenesis is stimulated in the healing pedicle following dramatic changes of the blood flow conditions after ligation of major vascular branches that supplied the ovary. Thus a process of vascular network reorganization and growth is complicating the stages of local tissue repair. Interestingly, tumors transplanted to the ovarian pedicle after ovariectomy and myocardial infarctions, which form a very different pathophysiological setting, exhibit loops and segmentation similar to those detected in the present investigation. The latter are, however, less complex and some of them display pathological variants^16a (and Patan et al, unpublished data). Whereas in the healing pedicle one sprout-like structure was detected within a system composed of 16 loops, in the corresponding tumor transplant loops and sprout-like structures were detected in approximately equal proportion.^16a This difference could be the result of the existence of pathological variants of in situ loop formation and remodeling as well as segmentation that cause the formation of blind-ending tubes in the tumor, although we cannot entirely exclude the possibility of classical endothelial sprouting in this system.

In the present system, loop formation starts around day 3 in the wall of the dilated ovarian vein. Signs of segmentation are not yet visible. At the same time, PECAM-positive fibroblast-like cells that migrate in the forming granulation tissue toward vessel walls might contribute to the expansion of vessel diameters (online Figures 1A through 1D). This facilitates the division of the lumen by tissue folds and pillars in the processes of loop formation and segmentation. Around day 7, all stages of both mechanisms coexist in close proximity. They give rise to a very dense vascular network in the granulation tissue that exhibits early signs of regression around day 14 consisting of occlusion of vessel segments by invasion of macrophages and fibroblasts into their lumen (online Figure 1E). Continuation of this process results in the formation of less vascularized scar tissue around day 21.

Formation of Elementary Loops
The reconstruction of a large sequence of serial sections suggests, based on the architecture of the loop systems and their precursor stages, and on the overall absence of single sprouts or endothelial buds, the following steps for elementary loop formation. The vessel lumen evaginates around a part of the intervening vessel wall that forms a column of tissue and contains at least one core of an interstitial tissue structure (ITS) with specific ultrastructure.^6,8 This process results in formation of a free intraluminal ITS that is surrounded by an "elementary loop" composed of two sheath-like segments (the evaginations) that finally fuse to establish loop patency (Figures 7A and 7B). Fusion of the two segments requires thinning of the connection between the ITS and the lateral vessel wall to form a slender endothelial extension. Subsequent fusion of the opposing cell membranes in this extension causes formation of a transcellular hole that separates the ITS from the vessel wall.^6,8

View larger version (80K): [in this window] [in a new window]   Figure 7. Mechanisms of loop formation, growth (elongation), and remodeling in the wall of the large vein according to intussusception (from analysis and reconstruction of histological serial sections). A part of the vessel lumen is shown in red, the tissue of the wall that is remodeled in blue. Elementary loop formation in the wall of the ovarian vein by lumen expansion around an intervening part of the lateral vessel wall (A and B). Elementary loop remodeling by splitting of the central ITS to form compound loop systems parallel to the vessel wall (C through F) or away from it (G through J). Elementary loop growth occurs simultaneously and is achieved by elongation of the loop segments and growth of its ITS(s) (C through F and G through J); arrows indicate blood flow.

Concerning the molecular regulation of vascular morphogenesis, our present model is not well characterized. In classical subcutaneous wound healing, flt, one of the receptors of vascular endothelial growth factor, and mRNA for the tie receptor are expressed in endothelial cells of large ectatic vessels bordering the wound 3 days after wounding. On day 7, after the onset of angiogenesis, flt and tie mRNA are also expressed in the small vessels of the neovasculature that course throughout the wound.^21,22 This spatial and chronological pattern matches with our morphological data in the healing pedicle.

Loop Remodeling
Loop elongation is achieved by a simple increase in the dimensions of the central ITS and elongation of the loop that is surrounding it (compare Figures 2 through 5 and 7).

To form a mature vascular network that supplies the newly formed tissue filling the wound gap, the elementary loops not only elongate but simultaneously generate and rearrange new segments. The analysis of elementary loops 1, 2 (Figure 2), and (3) (Figure 3) reveals that loop remodeling by splitting of the central ITS can start when the loop is still at the stage of an elementary loop. Splitting of the central ITS forms a new segment that automatically reconnects to either the main vein (Figures 2 and 7C through 7F), or it connects both segments of the loop (Figures 3 and 7G through 7J). Based on this mechanism, additional connecting segments that supplement the elementary loop to form a compound loop system are generated.

Depending on the orientation of ITS splitting, the network either expands parallel to the main vein (Figures 7C through 7F) or away from it (Figures 7G through 7J). The relatively simple compound loop system formed by loops 1, 2, and (3) illustrates these initial steps of loop formation and remodeling. Our analysis of ITS division in development demonstrates that this process requires the existence of two cores within the ITS between which the endothelial cell layer can retreat. This causes endothelial thinning and cell membrane fusion between both cores to finally split the ITS in two parts.⁸

Loop remodeling can also be implemented by addition of new elementary loops starting with formation of tissue folds that project from the central ITS or from the lateral wall of a loop segment into the lumen. Separation of the tip of the fold causes formation of a new small ITS surrounded by a tiny elementary loop that enlarges the size and increases the complexity of the loop system (Figures 4F and 4G).

Vessel Segmentation: A Mechanism of Remodeling of Preexisting Vascular Segments
Another form of IMG was identified in the main vein ahead of the region of loop formation, in numerous smaller veins located around the wound suture in the central region of tissue repair, and in a large branch of the ovarian artery. The vessel lumen is divided by slender tissue folds that connect to each other in its center forming intervascular walls (Figure 6). We also identified this mechanism that we term "segmentation" in tumor xenografts transplanted to the ovarian pedicle and in myocardial infarctions^16a (and Patan et al, unpublished data). A similar mechanism has recently been proposed in tumor ascites fluid.¹⁶ During segmentation, slender intervascular walls also give rise to ITSs and tissue pillars (Figure 6). This corresponds to pillar formation by splitting of intervascular walls, recently reported in the chicken chorioallantoic membrane,⁸ in the tumor microcirculation,^7,16a and in myocardial infarctions (Patan et al, unpublished data). Importantly, similar to neovascularization, in our infarct model (Patan et al, unpublished data), segmentation was also detected in a large branch of the ovarian artery.

Loop Formation and Vessel Segmentation Complement Each Other
Segmentation likely expands and remodels the preexisting vascular network; in contrast, loop formation in the wall of the main vein transforms a part of a large vessel into a dense network of tiny loops. The newly formed network of loops and the preexisting network become increasingly connected. This allows for redirection of blood flow, which is initially oriented to supply the loop systems from the site of the large vein after having passed through the preexisting tissue. After leaving the arterial side, blood flow will now enter the loop systems through the small preexisting veins and finally leave the organ through the large vein. Further loop remodeling and continuous segmentation, occurring also on the arterial side, will give rise to a new microcirculatory system developing at the site of the wound that is connected to the preexisting, surrounding network. A normal perfusion pattern is subsequently reestablished that enhances the integration of the newly formed tissue into the organ context.

	Acknowledgments

 
This study was supported by grants PA 638/1-1 from the German Research Foundation (S.P.) and R35-CA56591 from the NIH (R.K.J.). We thank Max Patan for help with the graphical artwork.

Received March 29, 2001; revision received July 17, 2001; accepted August 20, 2001.

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