An Opus on the Science of Neovascularization

The 20th century saw great advancement in the diagnosis and treatment of macrovascular disease. From Herrick’s description of myocardial infarction in 1912¹ to the advent of angiography, thrombolysis, angioplasty, bypass surgery, and intravascular stenting the 1900’s saw a revolution in the understanding of vascular disease, to the great benefit of patients.

The latter part of the last century also saw the birth of our appreciation of the microcirculation. Starting with Judah Folkman’s documentation of the role of angiogenesis in tumor expansion² other disciplines have advanced the understanding of the role of the microcirculation, in many cases to the point of successful therapeutic intervention. Although Folkman’s initial claims regarding the role angiogenesis in cancer were met with harsh skepticism, if not derision, anti-angiogenic therapies are now a routine part of cancer care. Similarly the management of pathological angiogenesis in the eye with anti-angiogenic drugs has literally restored sight in thousands of patients.

The role of the microcirculation in cardiovascular conditions has come into greater focus only in the last few decades. In myocardial infarction, stroke, peripheral arterial and kidney disease the link between microvascular attrition or dysfunction and worsening physiology has gradually become well established clinically. The growing clinical awareness of the role of the microcirculation in disease pathophysiology has naturally led to attempts at therapeutic intervention, the development of which relies on non-clinical methods and models.

Accordingly the publication of “State-of-the-Art Methods for Evaluation of Angiogenesis and Tissue Vascularization: A Scientific Statement from the American Heart Association” by Dimmeler et al, is a timely and welcome addition to the literature.

The Statement is well and logically organized, starting with in vitro assays, then developmental models, followed by adult models of vascularization and then sections on lymphangiogenesis, vascular permeability, microvascular functional testing and imaging, histology and a special section on the micro and macrocirculation in diabetes. Lastly there is an overview of pre-clinical models of angiogenesis as tools for therapeutic development. The adult section, which comprises the bulk of the Statement is nicely sub-divided to make this a useful and practical reference.

The description of in vitro assays is excellent, outlining the rationale for the use of certain models of endothelial cell behavior, the strengths of the methods and their limitations in terms of modeling in vivo physiology. This section well summarizes the state of the art in an appropriately succinct manner.

The section on developmental models is also superb. The first section describes the mouse embryonic hindbrain and postnatal retinal models in detail and contrasts their relative utility in studying sprouting angiogenesis. The models have become very elegant since the development of cre-lox technology that enables the introduction of gene knockouts at specific times during development such that the lethality of a global knockout is avoided. To the extent that this vast field of study could be distilled into one section it has been done nicely here with adequate references for the interested reader to pursue.

Next there are sections on avian (chick, quail) and aquatic (zebrafish, xenopus) models. All are nicely summarized, with the historical significance of the models providing a nice perspective overall. The greatest detail is provided for the zebrafish, which is still widely used as a tool of discovery and mechanistic exploration, owing to the ability to easily visualize vascular evolution in vivo.

The section on adult angiogenesis begins with a brief overview of placental angiogenesis, which is useful as it represents normal angiogenesis in the adult. Next is a section on wound healing models of angiogenesis which is well written, sufficiently detailed and quite circumspect on the merits and liabilities of the model. Particularly useful is a section of cautions to those using the model, giving readers the benefit of the author’s obvious experience and healthy perspective. Many of the sections throughout the Statement provided this type of “counter-point” and these are great strengths, since they permit the transmission of distilled insights to the readers.

The section on Matrigel plug assays is detailed and thoughtful. There is great information about how the assay is read, what is measures, its limitations and how to put the information into perspective overall. Again the specific calling-out of limitations and caveats is very welcome.

I was glad to see a detailed section on tumor angiogenesis, given its historical importance and the emergence of the field of “onco-cardiology”. Although most researchers studying cardiovascular angiogenesis may not have occasion to employ a tumor model those that do will find this section to be excellent. Once again a degree of detail that can only be derived from experience is provided – what vessels should be counted? Where? How? Key questions, nicely laid out and answered for the reader.

The section on genetic models focuses on VEGF, which makes sense given its central importance to angiogenesis in pre- and post-natal life. The text and accompanying table provide a great overview of a vast field of study (a PubMed search of “vegf angiogenesis” yields 24,890 citations) as well as a pragmatic view of the state of the art.

The sections on hindlimb and coronary models are extensive. The hindlimb model has been widely used to evaluate new vessel formation and this section contains good detail on a variety of methods to assess the vasculature. Coronary ischemic models to study neovascularization have been employed primarily in small animals via acute ischemia. The section on coronary models contains remarkable detail on the methods and functional readouts.

The section on vascular pathology in diabetes is a nice addition. The evolution of human vascular abnormalities in diabetes is outlined, followed by a nice discussion of the animal models that have been used to study diabetic vasculopathy, their advantages and pitfalls.

The section summarizing pre-clinical models for therapeutic applications is an excellent distillation of the state of the art. It should be required reading for investigators and regulators who are confronted with the transition from pre-clinical to clinical studies. The authors succinctly note that the predictive power of pre-clinical models for therapeutic interventions designed to induce new vessel formation has been very poor. This does not mean that pre-clinical and laboratory testing is not valuable....far from it. The discovery, mechanistic investigation and proof-of-concept afforded by pre-clinical work will always set the stage for clinical studies, but the latter, judiciously approached, will always be the gold standard.

The role of the microcirculation in the setting of ischemic brain injury is well established, and the developmental aspect of CNS vascularization is nicely approached. Similarly, there is controversy regarding the role of progenitor cells in the formation of new vessels in adults. For example there are data from human studies that provide clear evidence of the role of circulating cells in vascular repair.^{3, 4} These data, generated by examining hearts from patients who had undergone sex-mismatched transplants, reveal that extracardiac cells contribute between 14% and ~40% of the endothelial cell population in the donor heart, with evidence that the contribution was greatest in the microcirculation and precipitated by injury signals.

Overall, this is a truly powerful piece that should sit on the shelf of any scientist working in this field, and maybe under the pillow of every graduate student and post-doc.