Cerebral cavernous malformations (CCMs) present a relatively low prevalence (0.16-0.5%), accounting for 5-15% of all central nervous system vascular malformations (1-3). The disease is characterized as low-flow vascular malformations composed of blood-filled sinusoidal locules known as ‘caverns’. At the histological level, CCM is characterized by the lack of mural elements of mature vascular structures (3).

The major clinical presentations are epilepsy, headaches or focal neurological deficits; however 30% of the patients are asymptomatic or presents non-specific headache (4). During disease progression, the growth of vascular malformations is associated with recurrent hemorrhages (annual hemorrhage rate of 0.6-11%/patient/year) (3,5), which is considered to be a consequence of the immature vascular network constituting the CCM lesions (6). In the management of this pathology, it is also important to take into account changes in arterial pressure, which can alter the hemorrhage propensity and patterns (7). Depending on the anatomical localization of the CCM, patient management relies on surgical resection, observation and symptomatic treatment. In surgical inaccessible lesions, drugs such as statins, anti-angiogenic agents or vitamin D3 have been tested, although none have revealed clear benefits (8-11).

The beneficial use of propranolol in childhood hemangioma, a close pathological counterpart of cavernous malformations, supports the putative use of propranolol in the management of patients with symptomatic CCM (12). Propranolol administration in patients with CCM, although not commonly prescribed, has effectively been used in children and appears to play a protective role in the prevention of CCM-derived hemorrhaging in adults (13-18).

A common feature of propranolol-sensitive vascular tumors, such as hemangioma and CCM, is the distinctive expression of CD15-positive ‘vasculogenic zones’ (19-21). Of note, the in vitro gain of CD15 is followed by embryonic stem cell differentiation into endothelial cells (ECs) (22), indicating the putative involvement of endothelial progenitor cells (EPCs) in the development of vascular malformations. The association between CD15-positive cells and neo-vessels formation is not novel; it was described, >40 years ago in immature vessels of the placenta (23,24); the effects of propranolol on the placental regression have also long been described (25).

The identification of EPC subsets in peripheral blood (PB) is not yet clear. The authors have previously demonstrated that in tumors and normal tissues, some ECs simultaneously express CD14 (monocytic marker) and CD31 (EC marker), indicating mixed features between monocytes and ECs (26). More recently, it was demonstrated that monocytes can differentiate into ECs and be incorporated into blood vessels (27). These studies indicate the underestimation of monocytes as a relevant source for vascular growth. In fact, due to their 2-10% prevalence in PB (28) and compared to the estimated 0.002% of EPCs proposed by other studies (29-32), monocytes are putatively the most representative EPC subgroup in PB. Moreover, some studies have demonstrated that CD15 is also expressed in monocytes (33,34), with its levels being increased in pathological conditions (35). Notably, in tumors, CD14 immune cells are also CD15-positive, clearly indicating a subset of monocytes/macrophages (36). These observations indicate that CD15 cells in the ‘vasculogenic zone’ are in fact monocytes functioning as EPCs, contributing for blood vessel formation in CCM.

Hemangiomas and CCM share phenotypical characteristics, being both composed of a mixture of abnormal dilated capillary vessels with disorganized ECs and pericytes (37-39). The exact mechanisms that regulate the development of vascular abnormalities remain poorly understood. It is known that during the growth phase of hemangiomas, the increased expression of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) is associated with ECs and interstitial cell proliferation (37). The effect of propranolol in the reversion of vascular malformations is putatively associated with a decreased expression of FGF and VEGF, impairing EC migration, proliferation and reorganization, which in turns leads to vasoconstriction (involution phase) (12,40-42).

Since the natural evolution of CCM is chronic and unpredictable, the follow-up of patients with CCM involves the long-term clinical and imagiological evaluation with magnetic resonance imaging (MRI) (12). In an attempt to identify a suitable follow-up method, the monitorization of the levels of CD14⁺/CD31⁺ monocytes in the PB of patients with CCM is proposed. Considering that the authors recently published a study demonstrating that monocytes are viable EPCs (27), it was hypothesized that circulating CD14⁺/CD31⁺ monocytes function as EPCs and contribute to the development of CCM lesions.

Propranolol is a non-selective _β-adrenergic blocker commonly used in the control of anxiety and cardiovascular conditions, such as hypertension, myocardial infarction and angina pectoris. Over the past decade, propranolol was re-discovered as an effective drug in the treatment of certain vascular tumors, inducing the rapid involution to quiescent residual lesions in 80% of cases (12,47-50). Its use in the treatment of infantile hemangiomas, the most common benign tumor of the skin, has been discovered accidentally and it was verified that propranolol administration is highly efficient in inducing tumor regression with very few adverse effects (40). Thus far, the beneficial effects of propranolol have been observed in the treatment of neonatal hemangiomatosis (51,52), placental chorioangioma (53) and CCM (13).

In the present study, the administration of propranolol decreased the percentage of double-positive CD14⁺/CD31⁺ cells (monocytes) in the PB of the patient with CCM, reaching the levels presented by healthy blood donors during the follow-up period (Fig. 2A and B). The observed normalization of the CD14⁺/CD31⁺ cell levels upon propranolol administration suggested that the levels of circulating cells, sharing monocytic and EC features, are involved in CCM pathogenesis and are propranolol-sensitive. Moreover, it was hypothesized that circulating monocytes sharing EC features (CD14⁺/CD31⁺) function as EPCs, as was recently described (27), contributing to CCM progression by being incorporated into CCM neovessels.

The exact mechanisms through which propranolol interferes with angiogenesis are not yet known; however, some studies have indicated that its anti-angiogenic effects are mediated by the downregulation of VEGF and FGF levels (12,40-42). The dynamics of VEGF were also addressed in the present study, in an attempt to clarify whether the VEGF levels are linked to CCM regression. In the patient described herein, a decrease in the levels of VEGF in the PB was observed upon propranolol treatment (Fig. 2C); the levels were similar to the values observed in healthy donors (Fig. 2C). Notably, in vitro, monocytes appear to use more VEGF upon H₂O₂ exposure, decreasing its free levels in conditioned culture medium; however, a longer exposure to propranolol, rescued the observed decrease in VEGF levels due to H₂O₂ exposure (Fig. 3). This observation suggests that propranolol, apart from affecting the levels of circulating VEGF, can also affect the way monocytes use VEGF in vitro, thus decreasing the overall pro-angiogenic capacity. According to the decreased stimulation of monocyte differentiation into ECs, it was also observed that propranolol affected the proliferation (Fig. 4A) and migration (Fig. 4B) of mature ECs. These observations are in agreement with recently published data by the authors demonstrating that propranolol also impairs vessel-like structure formation by ECs (43). Accordingly, the exposure to propranolol disrupted vessel-like sprouting in aortic rings (Fig. 4C and D). Since the VEGF levels may also be involved in monocyte recruitment (54-56), the decreased levels of VEGF, upon propranolol treatment, may be responsible, at least in part, by the decrease in the levels of circulating CD14⁺/CD31⁺ cells and CCM regression. However, further studies are required to elucidate the mechanisms through which propranolol affects VEGF dynamics in monocytes and ECs.

In zebrafish, the lack of CCM1, 2 or 3 constitutes them a reliable CCM animal model, since it results in abnormal EC sprouting and thin-walled vessels (46,57). Therefore, through a murine and an embryonic zebrafish model, Li et al (58) demonstrated that propranolol ameliorated cavernous malformation, possibly through the inhibition of _β1-adrenergic receptor, once the silencing of this receptor prevented vascular abnormalities. Additionally, several research groups have already demonstrated that VEGF levels are regulated by the catecholamines' pathway, since its levels are proportional to _β-adrenergic receptors expression and can be inhibited by _β-adrenergic receptor antagonists (59-61). For instance, melanoma cell lines exposed to norepinephrine, an adrenergic receptor agonist, have been shown to exhibit increased VEGF levels (62). However, other studies have yielded contradictory results, demonstrating that the anti-angiogenic effect of propranolol is independent of its _β-blocker action. Sasaki et al (63) demonstrated that both _β blockade by active S(-)- and inactive R(+)-propranolol enantiomers were able to downregulate the expression of angiopoietin like 4, an angiogenesis regulator, leading to the impairment of hemangioma growth in vitro. In fact, besides its effects on differentiated cells, Seebauer et al (64) demonstrated that the treatment of a murine xenograft model with the R(+) enantiomer inhibited the differentiation of hemangioma stem cells to ECs and further vessel formation. Moreover, recently, the authors demonstrated that propranolol exerted an anti-angiogenic effect through an antioxidant mechanism accounting for the inhibition of a ferroptosis-like mechanism, which in turn, impaired EC activation and the formation of vessel-like structures (43). Therefore, propranolol may present diverse mechanisms of action to impair vascular growth.

In summary, propranolol, apart from promoting the regression of CCM, impairs CD14⁺/CD31⁺ cell circulation (Fig. 2A), in part by the decreased VEGF levels (Fig. 2C). It was also observed in vitro, that stimulation with a prooxidant (H₂O₂) tended to promote the differentiation of monocytes in cell culture medium towards ECs (27), with decreased levels of VEGF. The underlying mechanism may involve the control of monocyte differentiation into ECs and how these cells are phenotypically altered. As recently demonstrated by the authors, oxidative stress promotes monocyte differentiation into ECs, and this process is reversed by propranolol, which appears to attenuate oxidative stress (43). Furthermore, VEGF is essential during the differentiation of monocytes into ECs (27); however, when monocytes differentiate into macrophage-like cells, they become VEGF-producing cells (56,65). This switch from macrophages to ECs explains the dynamics of VEGF in cell culture media. Considering that monocytes functioning as EPCs may favor the development of CCM lesions and given that VEGF is pivotal for monocytic differentiation into ECs, the increased circulating levels of VEGF observed in the patient with CCM without treatment may be crucial for potentiating the EC differentiation route and further, for preventing CCM pathogenesis.

Although the propranolol mechanisms of action in CCM are not yet fully understood, the lack of a better therapeutic option for patients with surgically inaccessible CCM and the notable responses in a few patients suggest that it may be of value to explore the exact efficacy of propranolol in the treatment of CCM, as well as the associated adverse side-effects (12). In accordance with this, randomized prospective clinical trials with propranolol vs. placebo/nothing groups [phase 1 trial NCT03523650, phase 2 trials NCT03474614 and NCT03589014(66)] are currently ongoing. The findings of the present study reinforce the use of propranolol in the clinical management of CCM and points out the monitorization levels of monocytes (CD14⁺/CD31⁺) and VEGF in PB as useful tools which may be used to predict treatment efficacy.