This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Augustin, H. G. Search for Related Content PubMed PubMed Citation Articles by Augustin, H. G. Pubmed/NCBI databases Substance via MeSH

(Circulation Research. 2001;89:645.)
© 2001 American Heart Association, Inc.

Editorials

Tubes, Branches, and Pillars

The Many Ways of Forming a New Vasculature

Hellmut G. Augustin

From the Department of Vascular Biology & Angiogenesis Research, Tumor Biology Center, Freiburg, Germany.

Correspondence to Hellmut G. Augustin, DVM, PhD, Department of Vascular Biology & Angiogenesis Research, Tumor Biology Center, Breisacher Str. 117, D-79108 Freiburg, Germany. E-mail augustin{at}angiogenese.de

Key Words: angiogenesis • vasculogenesis • intussusception • intussusceptive microvascular growth

The angiogenic cascade is getting increasingly complex. A few years ago, vasculogenesis and angiogenesis were considered as the primary mechanisms leading to the formation of new blood vessels. The original definition of vasculogenesis denotes the formation of a primary embryonic vascular network from in situ differentiating angioblastic cells.¹ In contrast, angiogenesis primarily referred to the sprouting of blood vessels from preexisting vessels.¹

Recent advances in the identification of molecules that regulate angiogenesis and vascular remodeling have shown that the simplistic model of an invading capillary sprout is not sufficient to appreciate the whole spectrum of morphogenic events that are required to form a neovascular network (Figure 1).^1–3 Undoubtedly, vascular endothelial growth factor (VEGF) acts at an early point in the hierarchical order of morphogenic events and probably fulfills all criteria to be considered as a master switch of the angiogenic cascade. In contrast, the angiopoietins and their receptor Tie-2 as well as the ephrins and their corresponding Eph receptors appear to act at a somewhat later stage of neovessel formation. These molecules orchestrate a number of related, yet functionally and molecularly not well understood, processes such as vessel assembly (network formation and formation of anastomoses), vessel maturation (recruitment of mural cells [pericytes and smooth muscle cells], and extracellular matrix assembly, pruning of the primary vascular bed), and acquisition of vessel identity (formation of arteries, capillaries, and veins)^3,4 (Figure 2). Lastly, the mechanisms of organotypic differentiation of the vascular tree (continuous endothelium, discontinuous endothelium, fenestrated endothelium) are not at all understood and the first molecules that govern subpopulation-specific vascular growth and differentiation are just being uncovered.^5,6

View larger version (60K): [in this window] [in a new window]   Figure 1. Change of paradigm. From sprouting angiogenesis to vascular morphogenesis. Basement membrane degradation, directed endothelial cell migration, and proliferation (left) were considered as the primary mechanisms of angiogenesis. Corresponding in vitro assays have greatly helped to uncover molecules and mechanisms of angiogenesis. Today, the complexity of the sequential processes leading to the formation of a mature vascular network is increasingly recognized. These involve mechanisms of vessel assembly (network formation and formation of anastomoses), vessel maturation (pericyte recruitment, extracellular matrix assembly, pruning of neovasculature), acquisition of vessel identity (arteries, capillaries, veins), and organotypic differentiation (continuous endothelia, discontinuous endothelia, fenestrated endothelia). Yet, experimental systems to study these steps are largely missing.

View larger version (143K): [in this window] [in a new window]   Figure 2. Hierarchical order of morphogenic events during embryonic and adult growth of blood vessels. The primary formation of blood vessels occurs through mechanisms of vasculogenesis (center top). Vasculogenesis refers to the formation of a vascular network from precursor cells (angioblasts). Embryonic vasculogenesis results from the in situ coalescence of mesodermal angioblastic cells to form a capillary plexus. In contrast, adult vasculogenesis is mechanistically different and is mediated by the distal recruitment of angioblastic cells from precursor cell compartments (bone marrow). The secondary level of vascular morphogenesis describes the angiogenic formation of blood vessels. Angiogenesis refers to the formation of vessels and vascular networks from preexisting vascular structures (top, outer compartment). This can occur through classical sprouting angiogenesis with formation of anastomoses (top right) or through mechanisms of nonsprouting angiogenesis (top left). Nonsprouting angiogenesis occurs through mechanisms of intussusceptive microvascular growth (IMG) focally inserting a tissue pillar or by longitudinal fold-like splitting of a vessel. Sprouting angiogenesis and intussusception contribute to an increasing complexity of a growing vascular network. The network assembles and matures, eventually allowing directional blood flow. Cellular and biomechanical factors appear to be involved in shaping vascular identity (ie, arteries, capillaries, and veins), although there is also developmental biological evidence indicating that arteriovenous fate determination may occur before the formation of arteries and veins. Lastly, microenvironmental cues (extracellular matrix, cell contacts, and organ-selective growth factors) regulate the organotypic differentiation of a neovascular tree with continuous, discontinuous, and fenestrated endothelia. In contrast to the formation and maturation of new blood vessels through vasculogenic and angiogenic mechanisms, vascular remodeling describes the adaptational reorganization of an existing mature vasculature. This may occur acutely (eg, after sudden ischemia) or as a response to chronic stimuli (eg, atherosclerotic changes of vessel wall or in response to hypertensive biomechanical forces). The term "arteriogenesis" has been coined to describe the formation of collaterals from a preexisting capillary network after sudden ischemia as it occurs after cardiac ischemia or experimentally during surgically induced hindlimb ischemia. This process describes an adaptational remodeling phenomenon and should not be confused with the developmental acquisition of vessel identity that is associated with the formation of arteries, capillaries, and veins. Likewise, vessel cooption18 describes a vascular remodeling phenomenon originating from an existing vasculature that may contribute to tumor vascularization.

The function of these molecules has largely been elucidated through genetic experiments in mice ablating or overexpressing individual molecules. Yet, a detailed understanding of their molecular and functional mode of action is missing, which is primarily due to the lack of appropriate in vivo and in vitro models in which to functionally study these molecules. Most in vitro assays have very reductionist readouts such as endothelial cell chemoinvasion, migration, or proliferation. These assays have proven powerful in the early days of angiogenesis research. Yet, they are of limited use for the study of complex cellular interaction phenomena as they are associated with vessel assembly and vessel maturation. Likewise, most in vivo assays are either not quantitative or they are restricted to endpoint readouts that do not allow an appreciation of the dynamic three-dimensional spatiotemporal order of angiogenic processes, eg, in tumor models or in cardiac ischemia models. Most importantly, when it comes to studying angiogenesis in vivo, few laboratories apply three-dimensional techniques such as intravital microscopy or corrosion casting techniques to assess a neovascular bed. Instead, two-dimensional histological techniques are widely used to analyze angiogenesis in vivo. In fact, the counting of immunohistochemically stained microvessels in tissue specimens including tumors has become the gold standard to assess a neovascular bed in a given tissue.⁷ Clearly, this reductionist analytical approach is by no means sufficient to realistically reflect the whole spectrum of three-dimensional morphogenic events dynamically over time.

It is primarily a consequence of the limited availability of appropriate experimental models and analytical tools that vascular network formation through the process of intussusception is still not widely appreciated. Intussusception or intussusceptive microvascular growth (IMG) describes the formation of a vascular network from an endothelial cell–lined vessel by focally inserting a tissue pillar or by longitudinal fold-like splitting of a vessel. As a consequence, IMG can result in complex vascular networks by a nonsprouting angiogenesis mechanism.^1,2

The concept of vascular network formation through IMG is not new. Originally described more than 50 years ago,⁸ analytical work on IMG was pioneered in the late 1980s and early 1990s by the Swiss anatomist Dr P.H. Burri.^9,10 This early work has clearly shown that IMG is an important nonsprouting angiogenesis mechanism that contributes to capillary network formation independent of classical sprouting angiogenesis (Figure 2). Physiologically, IMG occurs in a number of embryonic and adult tissues, most notably during embryonic vascularization of the lungs¹¹ as well as during the cyclic changes of the endometrial vasculature in the adult.¹²

In two studies published in this issue of Circulation Research, Dr Patan and colleagues^13,14 have shed further light into the complexity of intussusceptive microvascular growth. They have used the isolated mouse ovarian pedicle model to study IMG during wound healing–like granulation tissue formation and during growth of tumors grafted onto the ovarian pedicle. In this model, the ovarian vascular supply is surgically manipulated so that the isolated ovary is at the end of a pedicle that is supplied by the ovarian artery and the ovarian vein. This model was originally developed to perform hemodynamic studies in an experimental tumor that is supplied by a single feeding artery and a single collecting vein.¹⁵ Patan et al have used this model to characterize the intussusceptive morphogenic remodeling of the ovarian vein and artery feeding into the granulation tissue¹³ as well as into LS174T human colon adenocarcinoma growing in the isolated pedicle.¹⁴ A zone of several millimeters was analyzed in both models through a carefully performed rather meticulous morphological analysis of several thousands of 2-µm serial sections. Computer-aided image analysis was then applied to three dimensionally reconstruct the vascular network. The results of both studies show quite clearly that IMG can lead to complex vascular networks completely independent of sprouting angiogenesis. Furthermore, the authors’ high-resolution approach demonstrates how intussusceptive vascular folds organize to establish compound loop systems resulting from tissue segmentation and intussusceptive anastomoses.

As with any intriguing study, the experiments by Patan et al raise numerous additional questions. For example, what are the driving forces behind IMG? There is some evidence that the angiopoietin/Tie-2 ligand/receptor system is involved in controlling IMG.^16,17 Likewise, biomechanical forces may be involved in regulating IMG. The surgical manipulation in the ovarian pedicle procedure used by Patan et al^13,14 leads to significant changes in hemodynamic forces that may be involved in remodeling the preexisting ovarian vein as much as hemodynamic forces are believed to act as critical regulators of collateral formation following cardiac ischemia (arteriogenic vascular remodeling). This also raises the question of a zonal analysis of the observed intussusceptive morphogenic events, ie, does IMG occur in the center or in the periphery of the analyzed granulation tissue¹³ and tumors?¹⁴ Zonal analyses of vascular morphogenic processes are particularly relevant in the context of tumor angiogenesis. Microvessel counting studies usually quantitate intratumoral microvessel densities. Yet, the tumor periphery marks the invasive zone of a tumor and gives rise to metastatic cell dissemination. Thus, the equilibrium between tumor angiogenesis and remodeling of the preexisting vasculature in the tumor periphery (vessel cooption)¹⁸ may be very relevant in determining tumor fate. Lastly, and possibly most important, what is the quantitative contribution of IMG (and the other mechanisms of vessel formation) to neovascularization and particularly to tumor vascularization?

It is increasingly recognized that vascular morphogenesis is a complex process driven by a number of different mechanisms that can lead to the formation of endothelial cell–lined blood vessels. Figure 2 summarizes the hierarchical order of our present understanding of hemangiogenic morphogenic events (as opposed to lymphangiogenic processes). Future work in the field of angiogenesis research will need additional tools and models to systematically analyze angiogenic processes to fully understand the complexity of the angiogenic cascade. This will also include the implementation of more sophisticated invasive and noninvasive techniques to analyze the vasculature of human tumors. The elegant, yet cumbersome experimental, approach taken by Patan et al^13,14 clearly reflects our limited ability to appreciate angiogenesis as a dynamic three-dimensional process. The implementation of analytical techniques that systematically assess human tumor angiogenesis beyond the counting of microvessel densities is just at its beginning.¹⁹ At the same time, novel angiogenic factors with a narrow cell and organ selectivity are being identified as inducers and modifiers of the angiogenic cascade.^5,6 Collectively, these observations indicate that the angiogenic cascade is far from being understood. Yet, a thorough understanding of the mechanisms of vascular morphogenesis will be a requisite for the rational translation of this knowledge into clinical application.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

1. Risau W. Mechanisms of angiogenesis. Nature. 1997; 386: 671–674.[Medline] [Order article via Infotrieve]

2. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000; 6: 389–395.[Medline] [Order article via Infotrieve]

3. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000; 407: 249–257.[Medline] [Order article via Infotrieve]

4. Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature. 2000; 407: 242–248.[Medline] [Order article via Infotrieve]

5. LeCouter J, Kowalski J, Foster J, Hass P, Zhang Z, Dillard-Telm L, Frantz G, Rangell L, DeGuzman L, Keller GA, Peale F, Gurney A, Hillan KJ, Ferrara N. Identification of an angiogenic mitogen selective for endocrine gland endothelium. Nature. 2001; 412: 877–884.[Medline] [Order article via Infotrieve]

6. Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K, Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishikawa S, Kishimoto T, Nagasawa T. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature. 1998; 393: 591–594.[Medline] [Order article via Infotrieve]

7. Fox SB. Tumour angiogenesis and prognosis. Histopathology. 1997; 30: 294–301.[Medline] [Order article via Infotrieve]

8. Short RHD. Alveolar epithelium in relation to growth of the lung. Philos Trans R Soc Lond B. 1950: 235: 35–87.

9. Caduff JH, Fischer LC, Burri PH. Scanning electron microscope study of the developing microvasculature in the postnatal rat lung. Anat Rec. 1986; 216: 154–164.[Medline] [Order article via Infotrieve]

10. Burri PH, Tarek MR. A novel mechanism of capillary growth in the rat pulmonary microcirculation. Anat Rec. 1990; 228: 35–45.[Medline] [Order article via Infotrieve]

11. Djonov V, Schmid M, Tschanz SA, Burri PH. Intussusceptive angiogenesis. Its role in embryonic vascular network formation. Circ Res. 2000; 86: 286–292.[Abstract/Free Full Text]

12. Rogers PA, Lederman F, Taylor N. Endometrial microvascular growth in normal and dysfunctional states. Hum Reprod Update. 1998; 4: 503–508.[Abstract/Free Full Text]

13. Patan S, Munn LL, Tanda S, Roberge S, Jain RK, Jones RC. Vascular morphogenesis and remodeling in a model of tissue repair: blood vessel formation and growth in the ovarian pedicle after ovariectomy. Circ Res. 2001; 89: 723–731.[Abstract/Free Full Text]

14. Patan S, Tanda S, Roberge S, Jones RC, Jain RK, Munn LL. Vascular morphogenesis and remodeling in a human tumor xenograft: blood vessel formation and growth after ovariectomy and tumor implantation. Circ Res. 2001; 89: 732–739.[Abstract/Free Full Text]

15. Gullino PM, Grantham FH. Studies on the exchange of fluids between host and tumor, I: a method for growing "tissue-isolated" tumors in laboratory animals. J Natl Acad Inst. 1961; 27: 679–693.

16. Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maguire M, Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature. 1995; 376: 70–74.[Medline] [Order article via Infotrieve]

17. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996; 87: 1171–1180.[Medline] [Order article via Infotrieve]

18. Holash J, Maisonpierre PC, Compton D, Boland P, Alexander CR, Zagzag D, Yancopoulos GD, Wiegand SJ. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science. 1999; 284: 1994–1998.[Abstract/Free Full Text]

19. Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res. 2000; 60: 1388–1393.[Abstract/Free Full Text]

This article has been cited by other articles:

				G. Murphy, N. Caplice, and M. Molloy Fractalkine in rheumatoid arthritis: a review to date Rheumatology, October 1, 2008; 47(10): 1446 - 1451. [Abstract] [Full Text] [PDF]

				B. Larrivee, C. Freitas, M. Trombe, X. Lv, B. DeLafarge, L. Yuan, K. Bouvree, C. Breant, R. Del Toro, N. Brechot, et al. Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis Genes & Dev., October 1, 2007; 21(19): 2433 - 2447. [Abstract] [Full Text] [PDF]

				D. McLeod A chronic grey matter penumbra, lateral microvascular intussusception and venous peduncular avulsion underlie diabetic vitreous haemorrhage Br J Ophthalmol, May 1, 2007; 91(5): 677 - 689. [Abstract] [Full Text] [PDF]

				O. Baum, L. Da Silva-Azevedo, G. Willerding, A. Wockel, G. Planitzer, R. Gossrau, A. R. Pries, and A. Zakrzewicz Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2300 - H2308. [Abstract] [Full Text] [PDF]

				G M Tozer Measuring tumour vascular response to antivascular and antiangiogenic drugs Br. J. Radiol., December 1, 2003; 76(suppl_1): S23 - S35. [Abstract] [Full Text] [PDF]

				L. Zhang, N. Yang, J.-R. Conejo-Garcia, D. Katsaros, A. Mohamed-Hadley, S. Fracchioli, K. Schlienger, A. Toll, B. Levine, S. C. Rubin, et al. Expression of Endocrine Gland-derived Vascular Endothelial Growth Factor in Ovarian Carcinoma Clin. Cancer Res., January 1, 2003; 9(1): 264 - 272. [Abstract] [Full Text] [PDF]

This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Augustin, H. G. Search for Related Content PubMed PubMed Citation Articles by Augustin, H. G. Pubmed/NCBI databases Substance via MeSH