Introduction

Until about 20 years ago, with blood doping and erythropoietin, cheating endurance athletes had a head start in the cat-and-mouse game with the World Anti-Doping Agency (WADA). In their efforts, the cardiovascular system and its oxygen-transport capacity have been central targets for manipulation. It is generally believed that the introduction of the Athlete Biological Passport (ABP) has largely kept the use of blood doping and erythropoietin at bay [1]. However, a new front is opening up: next-generation doping. We use this term to collectively describe the use of recent advances in medicine, such as gene therapy, gene editing, and targeted drug delivery, for doping purposes. As molecular biology and gene therapy technologies improve, the risk of misuse to enhance athletic performance in both human and non-human animal athletes increases [2–5]. WADA predicted this over 20 years ago, when, in 2003, it added gene doping to its list of prohibited substances and methods, responding to the rapid development in this area [6].

While some form of doping has been used by elite athletes since ancient Greece and Rome [7], doping opportunities have been on the rise, facilitated by the discovery and development of effective drugs to treat diseases. With the arrival of unprecedentedly powerful new drug classes within the last decades (recombinant proteins, targeted drug delivery, gene therapy, and gene editing), doping has become a considerable liability to the integrity of sports [8].

To maximize the blood’s oxygen-carrying capacity, erythropoietin has famously been used to increase the red blood cell (RBC) count, and before that (and perhaps again increasingly) blood doping, which dates back to the early 1970s, when the first successful attempts were, by rumor and admission, made by Finnish long-distance runners [9]. Although there is likely substantial heterogeneity among athletes [10], the major bottleneck in top endurance athletes is believed to be the cardiovascular system’s ability to deliver oxygen (“flow-limited athletes”) [11, 12]. Stimulating red blood cell production with erythropoietin injections has received considerable attention due to its use by prominent cyclists [13]. In the context of endurance sports, public perception and doping control have focused on increases in hemoglobin and RBC density, as well as on concurrent hematological parameters [14, 15]. However, a substantial part of the increased ability to deliver oxygen to muscles that results from endurance training is attributed to an increase in total blood volume [16]. In line with this, the total circulating mass of hemoglobin displays a stronger relationship with maximum oxygen uptake than hemoglobin concentration or blood volume alone [16–18].

Endurance training increases red blood cell production. Yet, the majority of this increase is not visible as an increase in hemoglobin or RBC concentration, but rather is absorbed by the larger blood volume. An increase in blood volume inevitably requires an expansion of the intravascular compartment (the fluid-filled space within blood vessels) to accommodate the blood. Both blood composition and blood volume have been extensively researched, but the changes to the vascular space itself have been much less studied. The scarcity of studies in humans is not surprising since the study of blood vessels in skeletal muscles requires repeated invasive procedures that are prohibitive for competitive athletes. While erythropoietin acts primarily on the RBC production, prolyl hydroxylase domain (PHD) inhibitors induce a broader hypoxia-responsive transcriptional program. In addition to increasing erythropoietin expression, they upregulate genes of the VEGF family, most notably VEGFA, the central angiogenic growth factor. VEGF-A plays a key role in regulating capillary density by stimulating endothelial cell proliferation and migration. Endothelial cells form the inner lining of all blood vessels and constitute the critical interface between circulating blood and surrounding tissues [19]. Also, erythropoietin itself exerts effects beyond RBC formation, including direct actions on the vascular system [20]. However, to date, none of the published human clinical studies has examined the effect of PHD inhibitors on total blood volume or capillary density.

When increasing the total blood volume by transfusion, the body rapidly normalizes hematological parameters [21]. Although blood doping has repeatedly been shown to be effective (reviewed by [22]), the basis of this enhanced performance remains unclear [21]. The performance gains do not only result from changes in blood composition, but the blood vessels themselves also play a critical role, as their collective internal volume imposes constraints on the maximal blood volume, which limits in turn red blood cell numbers. Hence, the factors that regulate blood vessel growth—notably the VEGFs—have become particularly interesting in efforts to increase exercise performance, not least because they and their effects are relatively difficult to detect.

The vascular system is essential for athletic performance as it delivers the oxygen and nutrients needed for sustained exercise while removing CO2 and waste products. Endurance performance is primarily limited by the maximal rate of oxygen uptake (V̇O2max), which is governed by a series of convective and diffusive resistances. The convective pathway is defined by maximal cardiac output and arterial oxygen content, ensuring bulk delivery to the microcirculation. The diffusive pathway is determined by the oxygen pressure gradient between the capillary and the mitochondria and the muscle's diffusing capacity (see Fig. 1) [27].

Fig. 1: Increasing oxygen availability has been a central goal of training and doping. For doping purposes, angiogenesis—the growth of new blood vessels—has received less attention than the composition of the blood itself. A Laminin staining in a cross-section of human skeletal muscle shows the basement membrane surrounding each myofiber and capillary. The partial pressure gradient of oxygen between the capillary and the mitochondria, together with the diffusion distance, is a primary determinant of the rate of oxygen flux in the terminal diffusion pathway. Image CC-licensed from [23]. B Angiogenic growth factors which mediate the physiological increase in vascular density resulting from training, can also be applied exogenously. To assess the angiogenic effect of VEGF-A, the disc on the left was coated with VEGF-A and applied to the chorioallantoic membrane (CAM) of a developing chicken, resulting in a denser vascular network and more angiogenic sprouts. The CAM assay is frequently used to test the angiogenic potential of compounds [24, 25]. Analogous to sports performance, the CAM’s ability to deliver oxygen is a limiting factor for the speed of embryonic development [26]

Enhancing capillary density through angiogenesis is a fundamental physiological adaptation to exercise training because it increases the mean transit time of red blood cells and expands the surface area for exchange. A higher capillary density reduces diffusion distances and prevents the ‘functional shunting’ of oxygen at high flow rates, thereby optimizing the microenvironment for oxidative metabolism and elevating the ceiling for aerobically generated power [28].

Targeting oxygen transport has been proven to be a successful doping strategy in the past. The use of erythropoietin among cyclists was largely unchecked before WADA began testing in 2000, after piloting testing schemes since 1996. Still, despite testing, elaborate techniques were deployed to evade detection [29]. For example, erythropoietin was combined with intravenously administered volume expanders, such as hydroxyethyl starch (HES), which has been marketed under trade names such as Voluven® and is included on the WADA Prohibited List [30], to lower otherwise suspiciously elevated hemoglobin levels [31]. Manipulating VEGFs in a controlled manner to expand the blood volume the cardiovascular system can hold could increase its oxygen-carrying capacity without increasing hemoglobin levels or RBC concentration [8].

This narrative review examines angiogenic doping as an emerging threat to sports integrity. The aims are to (1) evaluate the biological basis for angiogenesis-based performance enhancement, focusing on VEGF family growth factors as the primary and direct regulators of angiogenesis and their transcriptional activators; (2) assess the feasibility and accessibility of such angiogenic doping methods, from pharmacological agents to gene therapy and gene editing; and (3) analyze current detection challenges and potential countermeasures. An initial systematic literature search (see Supplemental Fig. 1 in the electronic supplementary material [ESM]) revealed that angiogenic doping remains an understudied area. The initial draft was therefore enriched with expert contributions to corroborate the findings with evidence from vascular and molecular biology, exercise physiology, clinical trials, and anti-doping research. Unless explicitly specified otherwise or obvious from the context, the data referenced in this review are derived from human studies. In Sects. 2 through 3.1, we only indicate exceptions to the rule of thumb that basic biomedical research is performed in rodents if there are reasons to believe that significant species differences might exist.