Physiologically, AGT is continuously produced by the liver, and is differentially expressed in different tissue types including adipose tissue, heart and kidneys (38). Within the plasma, AGT is converted to angiotensin I (ATI) by renin – a closely regulated enzyme synthesized and released by the juxtaglomerular cells in the kidney. This is a rate limiting step for systemic ATII production (39) (Figure 1).

Renin is produced in an inactive form, which is first cleaved to form pro-renin which is released as an inactive precursor, or is converted by proteases, into intracellular renin which is stored within juxtaglomerular cells (40). Pro-renin binds to the pro-renin receptor (PRR), which involves Wnt signaling, the vacuolar H+ adenosine triphosphatase, Par3 and tyrosine-phosphorylation signaling pathways (41, 42). Renin is then released from granules with in juxtaglomerular cells once stimulated by various mechanisms (39, 43), including the ATII negative feedback (43).

ATI is converted to ATII by angiotensin-converting enzyme (ACE), also known as ACE₁ – a membrane bound exopeptidase. ACE also degrades vasodilating peptides such as bradykinin, Ang(1-7) and kallikrein, making it an important vasopressor enzyme (39, 44).

ATII exerts its effects via its G-coupled protein receptors – ATII receptor 1 (AT₁R) and ATII receptor 2 (AT₂R), resulting in short-term cellular effects, as well as long-term genetic effects (45). AT₁R signaling also trans-activates epidermal growth factor signaling in breast epithelial cells, resulting in activation of the Ras/RAF/MAPK/ERK pathway, which drives cellular proliferation and cytoskeletal remodeling (46). ATII also causes production of aldosterone, a crucial regulator of blood pressure (47) implicated in oxidative stress and tissue fibrosis (48). Conversely, binding of ATII to AT₂R opposes the actions of AT₁R, although much remains unknown about this less investigated ATII receptor (49). Both AT₁R and AT₂R are expressed in most human tissues, with AT₁R being significantly more abundant in adult tissues, with the converse being true during fetal development (49).

ATII is converted to angiotensin III (ATIII), and then angiotensin IV (ATIV), by aminopeptidases (Figure 1). ATIII binds to AT₁R with less affinity than ATII, and conversely, AT₂R with more affinity than ATII. ATII is cleaved by angiotensin-converting enzyme 2 to produce Ang(1-7), which then binds to Mas receptors (MasRs). Ang(1-7) can also bind to AT₂R, and to Mas-related-G protein coupled receptors (MrgDs). Alamandine is the main ligand for MrgDs, which is formed by decarboxylation of Ang(1-7) (50). The Ang(1-7)/MasR axis is protective, by various mechanisms, which include inhibition of cellular proliferation and invasion, metastasis, and epithelial-to-mesenchymal transition (EMT), in several cancer types (51). However, pro-tumorigenic roles of the Ang(1-7)/MasR have been demonstrated in some cancers. For example, Ang(1-7)/MasR signaling stimulates the migration ability of cancer cells in renal cell carcinoma and renal cell adenocarcinoma (52). Interestingly, downstream Ang(1-7)/MasR signaling may be involved in platinum-resistance in non-small cell lung cancer, by inhibiting of angiogenesis and cancer growth (53).

As an endocrine cascade classically involved in the regulation of blood pressure and electrolyte homeostasis, the RAS is traditionally targeted using β-blockers, ACE inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) in the treatment of hypertension and other cardiovascular diseases (54).

The RAS has multiple bypass loops consisting of lysosomal proteases such as cathepsins B, D and G, and the mast cell protease chymase (Figure 1). Pro-renin is converted to renin by either cathepsin B or cathepsin D. Cathepsin D converts AGT to ATI. Cathepsin G converts AGT directly to ATII, or from ATI to ATII (3). Chymase converts ATI to ATII (55). These bypass loops are relevant, as the effect of traditional RAS inhibitors (RASIs) in blocking the RAS may be circumvented by the activation of these bypass loops, resulting in shunting of precursor peptides through for continued production of the downstream ATII.

In addition to acting systemically, there is increasing evidence of the RAS acting as a paracrine system in local tissues, as a key homeostatic “gatekeeper” broadly affecting multiple organ systems (38). Components of the RAS have been demonstrated in a wide-range of normal tissues (38), which act independently in an autocrine and paracrine fashion, and/or integrates with the systemic RAS, to exert its endocrine effects (56). A local RAS has been identified in the kidney (57), heart (58, 59), lung (60), brain (50), liver (61), pancreas (62), and is implicated in fibroproliferative and inflammatory conditions in specific organs, independent of the endocrine RAS (63).

A local RAS also operates within the bone marrow (BM) microenvironment, to regulate erythropoiesis, hematopoiesis, myelopoiesis, thrombopoiesis, and the formation of lymphocytic and monocytic lineages (64). The local RAS regulates primitive hematopoiesis in embryonic development, with the first hematopoietic and endothelial cells forming the marrow cavity in the embryo expressing ACE (64). Goker et al. (65) demonstrate that as well as influencing cellular proliferation across a host of tissues including the BM, RAS peptides significantly influence the pluripotential of hematopoietic stem cells. Haznedaroglu et al. (66) demonstrate that ATII utilizes the JAK/STAT pathway, which acts as a point of cross-talk between the local BM RAS and hematopoiesis. The local BM autocrine RAS is implicated in neoplastic hematopoiesis, with multiple RAS components being identified on leukemic blast cells (64). Renin is expressed on cells from acute myeloid leukemia, chronic myeloid leukemia, and acute lymphocytic leukemia (64) and its expression disappears following acute myeloid leukemia remission (67).

By interacting with the immune system, the RAS influences the TME which regulates resident ESC-like cells (5, 35), including CSCs (5). Aberrant RAS signaling is associated with organ dysfunction, and its role in these different groups of diseases is supported by both in vitro and in vivo studies (1, 3, 8) which highlight its fundamental homeostatic role in cell growth, migration and death, and inflammation (5, 68). Components of the RAS are present and are localized to ESC-like cells in different types of cancer (3–5, 34, 35, 68), VAs (1), and fibroproliferative conditions (8, 36).

The RAS influences the TME largely through its effects on the immune system, in particular, its role in creating immune-suppressive conditions (69). CSCs reside within the TME, which comprises various cellular and non-cellular constituents (70). One of the cellular elements - the endothelial cells, facilitate tumor development and protect tumor cells from the immune system (70). Also, endothelial progenitor cells within the TME contribute to angiogenesis from pre-existing vessels to foster tumor blood supply (71). Immune cells, such as macrophages, lymphocytes, and granulocytes, are another cellular constituent that contribute to an immunosuppressive TME to facilitate tumor cell survival (70). Extra-cellular matrix (ECM) is an essential non-cellular constituent of the TME, which consists of a network of glycoproteins, enzymes, and collagens. ECM also contains critical tissue components that regulate cellular proliferation, adhesion, communication between cells in the TME, and cancer cell migration (70). Further research is needed to understand how endothelial cells, immune cells, and the ECM confer a survival advantage to CSCs, and whether these interactions can be interrupted by inhibiting the RAS, which operates locally within the TME in different cancer types (5, 35). The RAS is expressed by various constituents of the TME, including CSCs, immune cells such as monocytes, macrophages, dendritic cells, neutrophils and T cells, with RAS signaling regulating these cells within the TME (69). The TME can promote or antagonize tumor growth, and modulates apoptosis, angiogenesis, cellular proliferation, metastasis, cancer-associated inflammation and desmoplasia (68, 69). The latter effects are generally driven by the ATII/AT₁R axis, with these pro-cancer effects being antagonized by the ATII/AT₂R and Ang(1-7)/MAS axes (69).

The role of the RAS in promoting cancer-associated inflammation and attracting tumor-promoting immune cells which enhance immunosuppression in the TME, is well documented (69). Furthermore, ATII/AT₁R axis signaling leads to hypoxia and acidosis within the TME, which increases the production of various inflammatory cytokines, transcription factors and the growth factors VEGF, hypoxia-inducible factor, and TGF-β. This creates an immunosuppressive TME, in which there are: (1) impaired function of T and dendritic cells, (2) buildup of immunosuppressive immune cells including myeloid derived suppressor cells, T_reg cells, and M2-like macrophages, and (3) elevated expression of PD-L1 and other immune checkpoint molecules that operate in cancer and immune cells (69). Treatment with RASIs may reduce the immunosuppressive effects of the RAS on the TME, which may confer improved treatment outcomes.

Taken together, the RAS – a fundamental homeostatic system – has far reaching effects systemically and locally in many tissue microenvironments, including the TME, with the immune system. Its effect on stem cells is in maintaining normal function of bone marrow stem cells, and in neoplastic hematopoiesis and other hematologic malignancies. We speculate that pathologic processes driven by the RAS may be modulated by RASIs and inhibitors of its bypass loops.

IH, the most common VA, affects 4–10% of infants (13). It is characterized by an initial rapid proliferation followed by spontaneous slow involution for up to 10 years, often leaving a fibrofatty residuum (13). Approximately 15% of IH require intervention during infancy due to a threat to function or life, tissue distortion, or ulceration (81). Problematic proliferating IHs were traditionally treated with high-dose corticosteroids, with interferon and vincristine indicated in refractory cases (82). Accelerated involution of IH induced by propranolol (a non-selective β-blocker) (83) and acebutolol (a selective β₁-blocker) (84) was serendipitously observed by two independent French groups. This led to a paradigm shift in the treatment of IH, with propranolol now being the mainstay treatment for problematic proliferating IH (Figure 3), although, many other β-blockers have been used (81, 85, 86), indicating a common mode of action by this class of drugs.

The proposed mechanisms of accelerated involution of proliferating IH induced by propranolol include inhibition of angiogenesis, vasoconstriction, and induction of apoptosis of proliferating endothelial cells (85), with mounting evidence implicating the RAS (13, 85, 87).

Research over the last two decades has demonstrated the expression of stemness-associated markers OCT4, stage-specific embryonic antigen-4 (SSEA-4), and STAT-3 on the microvessels of proliferating IH, and a stromal cell population expressing the stemness-associated markers NANOG (74), SALL4 (88), and CD133 (88). The expression of these markers decreases during involution of IH (74). The endothelium of proliferating IH also expresses the primitive mesoderm marker brachyury, hematopoietic markers including ACE (89), neural crest cell and neural crest stem cell markers (90), mesenchymal markers including vimentin and CD29, and the MSC marker Pref-1(13, 91), with a capability of undergoing spontaneous primitive erythropoiesis in vitro (13, 92). This phenotype and functional capability suggests the presence of a hemogenic endothelium phenotype (13, 89). Hemogenic endothelium gives rise to progenitor cells and hematopoietic stem cells in the yolk sac, aorta and placenta during embryonic development (93). There is also compelling evidence showing a placental origin of IH with this primitive endothelium sharing the phenotypic signature of placental chorionic villous mesenchymal core cells (13, 94).

The endothelium of proliferating IH expresses components of the RAS: ACE, AT₂R and PRR (76), with PRR being expressed by both endothelial and non-endothelial cells (95). Renin promotes endothelial cell proliferation in IH which is blocked by dickkopf-1 — a Wnt receptor blocker of Wnt signaling (96). This suggests there are interactions between renin, Wnt signaling, and the PRR (95) which is a part of the Wnt/frizzled receptor complex (97) (Figure 1).

RAS modulation influences cellular proliferation in IH, in vitro (1). Treatment of IH explants with an ACEI or AT₂R antagonist significantly reduces expression of Ki67, a marker of cellular proliferation. AT₂R agonism increases the proportion of Ki67⁺ cells, and treatment with the ACEI ramipril, prevents ATI-induced cellular proliferation in IH explants (98).

Plasma renin activity is highest in the first year of life, being 17-fold higher compared to adulthood, which then decreases rapidly to 8-fold of adult levels at 1–4 years of age, and then 5-fold at 5–9 years of age, followed by a tapering to normal levels in adults at 10–15 years old (99). The inverse correlation of serum levels of renin activity with age from birth, mirrors the programmed biologic behavior of IH (99–101). The important role of renin in fueling proliferation of IH is further supported by an increased incidence of IH in white, female, and premature infants, all of whom have elevated endogenous levels of renin, compared to their counterparts (87).

The involvement of the RAS in IH is further supported by the demonstration of accelerated involution of problematic proliferating IH induced by the ACEI captopril (102) (Figure 4). Serum levels of renin, ACE and ATII are reduced in patients following surgical excision and propranolol treatment (87). Further, the level of serum ATII decreases following administration of the ACEI captopril, of IH (87).

Cathepsins B and D which constitute bypass loops of the RAS are expressed by the endothelium of the microvessels and cells in the interstitium (103), and cathepsin G (103) and chymase (104, 105) by mast cells within the interstitium, of IH. These bypass loops may enable continued production of ATII despite blockade with RASIs, and offer a potential explanation for the variable response of IH to β-blockers (85, 103).

Propranolol is administered as a racemic mixture of R- and S-propranolol. S-propranolol is an active enantiomer, and R-propranolol an inactive non-β-blocking enantiomer (106). Recent evidence shows that R-propranolol inhibits the growth of bEnd.3 hemangioma cells in vivo, independent of β-blockade (107). This is interesting, as R-propranolol inhibits the transcription factor SOX18. SOX18 mutation causes hypotrichosis-lymphedema-telangiectasia and renal syndrome, which features lymphatic malformation. Propranolol as an effective long-term treatment for hypotrichosis-lymphedema-telangiectasia and renal syndrome, provides evidence it may cause accelerated involution of IH via a SOX18-dependent pathway, rather than just affecting the β-adrenergic receptors (106). These findings present an opportunity to investigate new therapeutic options for IH and other conditions using an enantiopure R(+) propranolol, instead of its conventional racemic mixture, allowing for a lower dosage and thus lower side effects of the treatment (106).

Components of the RAS are expressed by cells expressing stemness-associated markers in other types of VAs including pyogenic granuloma (77), VM (78), and LM (80).

The RAS regulates stem cells (108) and interacts, at multiple levels, with the Ras/BRAF/MEK/ERK and PI3KCA/AKT/mTOR pathways which regulate cellular proliferation, differentiation, and stem cell maintenance (1) (Figure 5). Multiple gain-of-function gene mutations affecting these pathways that cause over-activity, have been identified in a number of VAs (1, 94, 109–111). The central role of the RAS in regulating hematopoietic cells, MSCs and angiogenesis, and the presence of components of the RAS (77, 78, 80, 102) and its bypass loops on ESC-like cells in VAs (103, 112–114), suggests possible therapeutic targeting of the ESC-like cells with RASIs in VAs (1).

Treatment of patients with CLOVES (a PIK3CA-related overgrowth syndrome) with the PIK3CA inhibitor BYL719P leads to improved symptoms with a decrease in the size of their VAs, and improved cardiovascular measures such as congestive heart failure, cardiac hemihypertrophy, and scoliosis (115). Vemurafenib restores normal blood flow of AVM in a BRAF-mutated zebrafish model (116). Low-dose AKT inhibitor miransertib (ARQ092) reduces the levels of phospho-AKT by approximately half in 83% of tissue samples of patients with Proteus syndrome, caused by gain-of-function mutations in AKT in the PI3KCA/AKT/mTOR pathway (117). Targeted therapies that inhibit MAPK2K1/ERK for these mutations have been investigated in many cancer types, including metastatic MM (118), advanced soft tissue sarcoma (119), and ganglioglioma (120). Targeting mutations in MEK1/2 (MAP2K1) have also been proposed for VAs. The interaction between the PI3CK/AKT/mTOR and RAF/MEK/ERK pathways via Ras may enable bypass of single step blockades by targeted therapies. In non-small cell lung cancer, kras mutations result in activation of the RAF/MEK/ERK pathway, where by inhibition of MEK with a MEK inhibitor is ineffective because of alternative activation of the PI3CK/AKT/mTOR pathway (121). Furthermore, resistance to MEK inhibitors may be worsened by activating mutations in PI3CKA, and by a mutation in PTEN (which inhibits PIK3CA) causing complete resistance (122). The potential of resistance to targeted therapies due to activation of alternative pathways, highlights the importance of continuing a broad search for safe and effective treatment strategies for VAs (1).

Sirolimus (rapamycin), a mammalian target of rapamycin (mTOR) inhibitor with anti-angiogenic, anti-lymphangiogenic, and anti-proliferative properties (123, 124), is traditionally used as an immunosuppressant in organ transplantation to prevent organ rejection (125). It is increasingly used for complex VAs (126) and PIK3CA-related overgrowth syndromes (127). mTOR is regulated by PI3K, which is involved in many cellular processes, including cellular proliferation, metabolism, autophagy, growth, as well as angiogenesis and lymphangiogenesis (126). Since its first use in an infant with kaposiform hemangioendothelioma refractory to conventional therapies (128), several studies have investigated its efficacy and safety profile for VAs. A systematic review by Freixo et al. (126) concludes that sirolimus improves outcomes for VAs, particularly vascular tumors associated with life-threatening coagulopathy, as well as LM and VM. In total 95.5% of patients with kaposiform hemangioendothelioma improve clinically, with correction of coagulopathy in 93% of patients. Size reduction is observed in 88.9% of patients with VM, and clinical improvement is experienced by 94.9% of patients with LM, following sirolimus treatment. However, no benefit of sirolimus has been demonstrated in patients with AVM. Of the 162 patients with vascular tumors and 211 with vascular malformations treated with sirolimus, multiple side effects were observed, including oral mucositis (31.9%), dyslipidemia (16.5%), leukopenia (12.3%), gastrointestinal symptoms (10.2%), and rash/eczema (8.2%) (126).

Sirolimus is involved in regulating cell stemness, which is relevant given the recent observation of cells expressing stemness-associated markers in many types of VAs (1). Short courses of sirolimus enhance somatic cell reprogramming, and longer-course treatment and mTOR-knockout reduces programming (129). Further, mTOR activation in human somatic cells with ectopic expression of OCT4, SOX2, KLF4 and c-MYC, increases the production of iPSCs (130). Interestingly, inhibiting mTOR induces a paused pluripotent state in mouse blastocysts, but maintains their phenotypic signature and pluripotency, suggesting that mTOR regulates developmental timing at the peri-implantation stage (131). The observed efficacy of sirolimus in VAs and the known role of mTOR in regulating stem cells, suggest a possible effect of mTOR inhibition on the primitive population within VAs (1). Sirolimus may alter or inhibit the stemness of these transcription factors within VAs, depending on the dose and duration of treatment (1).

In conclusion, VAs are a heterogeneous group of conditions that remain largely unsolved. The recent discovery of stem cells and the RAS in IH underscores the programmed biologic behavior and accelerated involution of proliferating IH induced by β-blockers and ACEIs, and provides a novel line of inquiry into other VAs. The efficacy of sirolimus in complex VAs, and the presence of ESC-like populations in multiple types of VAs points to ESC-like populations being a potential therapeutic target. Given the presence of components of the RAS and its bypass loops, it is interesting to speculate that these primitive populations may be targeted using RASIs.

The CSC concept (Figure 6), also known as the hierarchical model of cancer, proposes that cancer progression is driven by CSCs (2). CSCs possess pluripotency and self-renewal properties, and are the proposed initiating cells and the driver of the tumor growth (2). CSCs are highly tumorigenic, divide symmetrically to form identical CSCs, and asymmetrically to form differentiated non-tumorigenic cancer cells that give rise to the bulk of the tumor (132). CSCs are attributed to tumor dormancy, metastasis (2), resistance to chemotherapy and radiotherapy (133) and immunotherapy (134), and recurrence (135).

CSCs have been demonstrated in many cancer types (2), including breast cancer (136), prostate cancer (137), glioblastoma (24), primary (22) and metastatic (26) colon adenocarcinoma, lung adenocarcinoma (23), oral cavity squamous cell carcinoma of different subsites (25, 28, 29), primary (30) and metastatic (12) HNcSCC, and metastatic MM to the regional nodes in the head and neck (31) and the brain (32).

Hypotheses on the origin of CSCs include de-differentiation of differentiated tissue somatic cells, or resident adult stem cells that become CSCs through genomic instability, an inflammatory tissue microenvironment, lateral gene transfer, and cell fusion (135).

Cancer metastasis is the main cause of cancer deaths (138). The “seed” and “soil” theory was first proposed in 1889, by which tumor cells (the seed) from the primary tumor travel to a distant organ (the soil) with a favorable environment for tumor cell colonization (139). Circulating tumor cells (CTCs) have been proposed to be responsible for metastasis (140). However, only a small proportion of CTCs possess metastatic potential, for example, 0.02% of melanoma cells injected into the portal circulation form macrometastases (141). CSCs are proposed to circulate in the circulatory system (142) and these may constitute the small fraction of CTCs that possess metastatic potential.

Cells within metastases phenotypically resemble CSCs (139). Tumor cells within a cancer undergo EMT to form mesenchymal-like cells that undergo intravasation to enter the blood and/or lymphatic circulations as CTCs. These CTCs that possess CSC characteristics undergo mesenchymal-to-epithelial transition (MET) and extravasate into distant tissue sites, where metastatic tumors may be established (Figure 7). MET is a fundamental process occurring during embryogenesis when the mesoderm becomes epithelial tissue in embryogenesis (143). CSCs may undergo EMT (144), which causes a phenotype conducive for tissue invasion and metastasis (145). It is interesting to speculate that a small proportion of CTCs have stemness properties, and a CSC-like phenotype, facilitating EMT and thus metastasis. EMT and MET play an important role in metastasis, which is intricately linked to epithelial plasticity (146). The role of CSCs in metastasis is supported by the demonstration that CD133⁺ and chemokine receptor CXCR4⁺ CSCs in pancreatic cancer have high metastatic potential, as do CD26⁺ CSCs in colon cancer (147, 148), and ALDH⁺ and CD44⁺/CD24⁻ CSCs in breast cancer (147).

Cutaneous SCC (cSCC) accounts for 15–25% of all skin cancers (149). The mainstay treatment of cSCC is surgical excision. About 2.6% of cases metastasize to the regional lymph nodes (150), with risk factors including tumor site, poor histologic differentiation, lymphovascular and/or perineural invasion (150), and tumor thickness and horizontal diameter (151, 152), and immunosuppression (153). The 5-year overall survival for metastatic HNcSCC is 48%, despite intensive treatment with surgery and adjuvant post-operative radiotherapy (154). These poor outcomes are attributed to the presence of CSCs (2, 12).

Populations of cells that express the stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC, have been demonstrated in primary HNcSCC (12) and metastatic HNcSCC (30). Metastatic HNcSCC-derived primary cell lines are capable of forming tumorspheres, providing preliminary evidence of ESC-like cells within metastatic HNcSCC possessing stem cell function (12). It has been recently shown that miR-22, an miRNA important for follicle stem cell differentiation, promotes cancer development and metastasis by maintaining β-catenin signaling and hence CSC function (155). The CSC subpopulations in both primary HNcSCC (156) and metastatic HNcSCC (33) express components of the RAS.

MM constitutes 1% of all skin cancers but has the highest mortality among skin cancers with 6,850 deaths in the United States in 2020 (157). Primary MM has a five-year survival of over 90% (158), but this decreases to 43% in patients with regional metastasis (159). MM can develop in other organs, including the mucosal surface of the nasal cavity, paranasal sinuses, and the upper aerodigestive tract, gastrointestinal tract, vulva, and the central nervous system (158, 160, 161).

Multiple gain-of-function mutations in the mitogen-activated protein kinase (MAPK) pathway are often present in MM (162), which have been treated with targeted therapies (163–165). Advanced MM is treated with immunotherapeutic agents, such as the monoclonal antibody checkpoints cytotoxic T-lymphocyte antigen-4 (CTLA-4) with ipilimumab, and programmed death 1 (PD-1) with nivolumab and pembrolizumab. These agents significantly improve the life expectancy of patients with advanced MM and those at high risk of recurrence (166). However, primary or acquired resistance to anti-PD-1 or anti-PD-1/anti-CTLA-4 remains a clinical challenge, and encourages researchers to continue looking for non-immunotherapeutics that may be integrated with immunotherapy (166).

Propranolol is associated with improved cancer survival outcomes in patients with head and neck, esophageal, stomach, colon, and prostate cancer (167). A recent phase II clinical trial demonstrates that 1 week of pre-operative treatment with propranolol reduces biomarkers for metastasis in breast cancer (168). It has been hypothesized that RAS modulators such as propranolol exert their anti-cancer effects via β-adrenergic signaling which enhances tumor growth via β₂-adrenergic receptors (169). A recent study shows that β₃-adrenergic blockade with SR59230A reduces tumor growth and significantly attenuates the stemness markers CD44, NANOG, OCT3/4 and CD24, implicating β-adrenergic receptors in maintaining the stemness of CSCs within the TME (170). Propranolol, a non-selective β₁- and β₂-adrenergic receptor antagonist, inhibits renin secretion. As the RAS is a stem cell regulator, the anti-cancer effect of propranolol may be the result of its action on the local RAS within the TME. RAS modulators has been shown to reduce pluripotency and stem cell capability of colon adenocarcinoma-derived cells that possess stem cell function (171).

CSCs that express stemness-associated markers have been widely documented in MM (172, 173), including cells expressing OCT4, SOX2, KLF4, c-MYC in metastatic MM to the regional lymph nodes in the head and neck (31) and the brain (32). These cells are capable of forming tumorspheres in vitro, providing preliminary evidence of stemness properties (31). CSCs in metastatic MM to the regional nodes in the head and neck (171) and the brain (174) express components of the RAS. Ishikane et al. (173) demonstrate the important influence of the RAS on MM metastasis. Two weeks following injection of B16/F10 mouse melanoma cells that do not express AT₁R, into C57BL/6 mice, the number of metastases is significantly greater in the group treated with ATII compared to the vehicle-treated group. The ARB valsartan, but not the calcium channel blocker amlodipine (which increases plasma renin levels), suppresses these effects of ATII. This data supports ATII in exacerbating hematogenous spread of MM by activating adhesion molecules in vascular endothelial cells. Renziehausen (175) demonstrates decreased expression and increased CpG island methylation of AGTR1 within metastatic MM, compared to primary MM, implying that AGTR1 acts as a tumor suppressor gene in MM. Furthermore, antagonizing AT₁R with an ARB or shRNA-mediated knockdown in melanoma cells lines expressing AGTR1, allows the cell lines to proliferate in serum-free conditions. Interestingly, treatment with the AT₂R antagonists PD123319 and EMA401 inhibits angiogenesis, melanoma cell growth, and increases the effect of BRAF and MEK inhibitors in cells with BRAF V600E mutations (175). The author proposes inhibition of AT₂R as a potential therapeutic option for treating MM which expresses the RAS.

Another potential therapeutic benefit of RASIs is their enhancement of the effectiveness of existing cancer therapies. For example, RASIs increase the effects of anti-PD-1 therapy on MM. Investigation of the role of the RAS in the MM TME by Nakamura et al. (174) demonstrates that fibroblasts produce CC motif chemokine ligand 5 (CCL5) upon stimulation of the RAS, which is suppressed upon ARB administration in mice, and that ARB administration (1) decreases CCL5 blood concentration, (2) increases tumor-infiltrating T cells, (3) decreases regulatory T cells, and (4) increases tumor antigen-specific T-cell responses. Furthermore, ARB and anti-PD-1 co-administration significantly improves tumor growth inhibition, compared with monotherapy. This highlights the potential of RASIs as an adjuvant treatment to improve the therapeutic efficacy of anti-PD-1 antibodies and highlights the interaction between the RAS and the immune system in the TME. Further work is warranted to investigate whether RASIs improve the efficacy of other targeted therapies.

In 1998, Lever et al. (176) first demonstrated that patients taking ACEIs and ARBs have a lower risk of several cancer types. Since then, there has been intensive investigation into the role of the RAS in cancer. The demonstration of components of the RAS in an increasing number of cancer types, and the emerging concept of a local tissue RAS and its associated non-classical roles, have led to the appreciation of the crucial role of the RAS in diseases including cancer, in addition to its classical endocrine effects (38).

Several observational epidemiological studies have shown a lower incidence of cancer and improved cancer outcomes in patients taking RASIs (3, 177) although some studies show no such association (41).

Propranolol increases the median progression-free survival and overall survival of patients with metastatic angiosarcoma by nine and 36 months, respectively (178). A systematic review and meta-analysis of 13 studies by Zhou et al. (179) demonstrates that administration of ACEIs or ARBs significantly improve survival outcomes in patients with gastrointestinal malignancies, with an improved overall survival (HR 0.79, 95%CI 0.70–0.89); p < 0.000), cancer-specific survival (HR 0.81, 95%CI 0.73–0.90; p < 0.000) and recurrence-free survival (HR 0.68, 95%CI 0.54–0.85; p = 0.001), but not progression-free survival (HR 0.88, 95%CI 0.73–1.07; p = 0.183) and disease-free survival (DFS; HR 0.50, 95%CI 0.11–2.39; p = 0.103). Another meta-analysis by Li et al. (180) demonstrates a significant improvement in overall survival (HR 0.80, 95%CI 0.69–0.92; p = 0.29) and progression-free survival (HR 0.79, 95%CI 0.66–0.94; p = 0.11) for those taking RASIs with chemotherapeutic agents, compared to those taking chemotherapy alone. A sub-group analysis demonstrates that platinum-based agents in conjunction with RASIs provides a significant benefit for pooled overall survival (HR = 0.56, 95%CI 0.38–0.82; p = 0.96) (180). A further meta-analysis of 55 studies by Sun et al. (177) also demonstrates significant improvement in overall survival (HR = 0.82, 95%CI 0.77–0.88; p = 0.001), disease-free survival (HR = 0.80, 95%CI 0.67–0.95; p = 0.01) and progression-free survival (HR = 0.74, 95%CI 0.66–0.84; p < 0.001) for those taking RASI.

Some randomized control trials (RCTs) show that RASIs have no effect on survival outcomes for cancer patients, and that some ARBs may increase cancer risk depending on the type of ARB. A decreased cancer incidence is associated with ARBs in observational studies (RR 0.91, 95%CI 0.84–.99; p = 0.022), however, there was no decrease in cancer incidence associated with ARBs in RCTs which was not statistically significant (RR 1.00, 95%CI 0.92–1.10; p = 0.964) (178). A recent meta-analysis of RCTs, which includes 68,402 patients with solid organ cancers from five trials, shows that ARBs are associated with a small increased risk of new cancer diagnosis. Patients randomly assigned to take ARBs have an increased risk of new cancer occurrence, compared with those in the control group (7.2 vs. 6.0%, RR 1.08, 95%CI 1.01–1.15, p = 0.016) (181). Furthermore, a protective effect of ACEIs on cancer development is not demonstrated (182), and another finds no association between ARB use and cancer risk (183). Lastly, ARB use has been associated with increased risk of lung cancer compared to patients in control groups (7.2 vs. 6.0%, risk ratio 1.08, 95% CI 1.01–1.15; p = 0.016) (181). The study design, the type of cancer studied, the duration of RAS agent administration, and the use of other medications are possible reasons for these conflicting results (184).

When analyzing epidemiological data on the effect of RASIs on cancer, ACE polymorphisms including the insertion/deletion (I/D), which influences the expression of ACE, should be considered. D/D carriers have higher ACE levels than I/I carriers, and the D/D polymorphism is associated with the extent of lymphatic metastases in gastric malignancy (179). D/D polymorphism has also been associated with worse prognosis in prostate cancer (185).

The RAS, its bypass loops and converging signaling pathways can be inhibited at multiple points (Figure 8) (3). For example, renin can be directly inhibited with the renin inhibitor aliskerin, and its secretion blocked by β-blockers such as propranolol. ACE can be inhibited by ACEIs, and AT₁R with ARBs. Cathepsins B, D, and G, and chymase, can be blocked by their respective inhibitors. Furthermore, signaling from AT₁R impacts the NOX/ROS/NFkB/COX2 axis, which may be targeted with NOX inhibitors, ROS inhibitors, metformin, and non-steroidal anti-inflammatory drugs, respectively. Other converging signaling pathways of the RAS that can be targeted include Wnt/β-catenin signaling, which is influenced by pro-renin, which may be targeted by Wnt/β-catenin inhibitors. Lastly, aminopeptidase A can be targted with aminopeptidase A inhibitors, and MasR with its antagonist A779.

The RAS may affect the expression of stemess-associated markers in cancer (186) and CSCs either directly or indirectly via its influence on the TME directly and by its interaction with the immune system (5, 35). Targeting the RAS, its bypass loops and converging signaling pathways to achieve optimal blockade of the RAS to regulate CSCs has been proposed (3–5). This “hollistic” approach of blocking the RAS at multiple points simultaneously, forms the basis of a phase I clinical trial for recurrent glioblastoma (187) resulting in a trend toward increased survival by 5.3 months. The encouraging results of this trial warrant further clinical trials on this novel, well-tolerated and cost-effective potential therapeutic option for patients with glioblastoma, and potentially other cancer types.

The growing interest in combined drug treatment for cancer is highlighted by the treatment of recurrent glioblastoma using the CUSP9^* protocol, where nine repurposed drugs are used simultaneously (188) with three of the 10 patients remaining progression-free at 63, 54 and 52 months following tumor recurrence (Halatsch et al., personal communication, December 21, 2021). Aprepitant, artesunate, auranofin, captopril, celecoxib, disulfiram, itraconazole, ritonivir, and setraline which are used in the CUSP9^*protocol, blocks one or more surival pathways used by glioblastoma cells, to potentially enhance the efficacy of temozolomide in the treatment of glioblastoma.

Taken together, the presence of CSCs – the proposed founding cells of cancer, and driver of metastasis, treatment resistance and recurrence, and the expression of componenets of the RAS and its bypass loops across multiple cancer types, raises the possibility of targeting CSCs with RASIs and inhibitors of RAS bypass loops.

KD is a fibroproliferative condition characterized by excessive dermal collagen deposition in response to skin injury, and/or inflammation (8). Keloid lesions (KLs) extend beyond the borders of the original wound, without spontaneous wound remodeling (190). KLs are rubbery plaque-like lesions that cause disfigurement, pruritis, tenderness, and functional problems such as joint contracture (191). KLs commonly occur on the earlobes, anterior chest, shoulders, and upper back, with increased sebaceous gland density and surface tension proposed as risk factors (8). Current treatments for KD include serial intralesional corticosteroid injections, and surgical excision with intra-operative steroid injections, cryotherapy, topical 5-fluorouracil, laser therapy, silicon occlusive dressing, and excision with post-operative radiotherapy (8).

ESC-like cells have been demonstrated in keloid-associated lymphoid tissues (KALTs) (20) consisting of aggregates of lymphoid cells expressing CD20, located in the reticular dermis, beneath the epidermis of KLs (192). This primitive population expresses the stemness-associated markers OCT4, SOX2, and pSTAT3 which are localized to the endothelium and perivascular cells of the KALTs (20). mRNA of the stemness-associated marker Rex-1 is also present in KLs, and SSEA-4 has been demonstrated in the reticular dermis of KLs (193) (Figure 9).

PRR, ACE, AT₁R and AT₂R are expressed on the ESC-like cells on the endothelium, with PRR also present on the pericyte layer, of the microvessels within the KALTs (194). Vitamin D receptor (VDR) is also expressed on the endothelium of the microvessels and perivascular cells within the KALTs (19). These ESC-like cells in KLs also express cathepsins B, D and G – proteases that act as bypass loops of the RAS (192).

Sustained activation of the RAS results in hypertension and organ fibrosis (195) via AT₁R signaling and an ATII-mediated increase in TGF-β (196). AT₁R signaling increases cellular proliferation, oxidative stress, inflammation, and creates a pro-fibroproliferative environment via inflammatory cell recruitment, and with other microenvironmental factors, ultimately resulting in excessive ECM deposition (197).

Dark-skinned individuals are predisposed to vitamin D deficiency (VDD) (198) which increases RAS activity via the genomic actions of VDR (199). The RAS alters the microenvironmental niche within KLs by influencing the immune system, the IL-6 and IL-17 axis, and by increasing vascular permeability and inflammation (8). Acting as a transcription factor, VDR signaling contributes to the formation of keloid fibroblasts and TGF-β/smad signaling, and may alter the expression of genes regulating endothelial-to-mesenchymal transition (endoMT) and EMT (8, 9, 200). Elevated RAS activity also increases ECM deposition and fibrosis via increased TGF-β signaling and increased fibroblast proliferation and migration (201). This complex network of interactions results in the accumulation of aberrant fibroblasts and myofibroblasts, via a MSC intermediate, from endoMT (8) (Figure 9). ATII mediates endoMT and RAS inhibition, with the ARB irbesartan preventing glucose-mediated increase in α-SMA in endoMT (202).

Dark-skinned individuals are predisposed to KD (203), VDD (204) and hypertension (205). The observation that VDD and hypertension are associated with elevated activity of the RAS (195, 199), and the expression of components of RAS by ESC-like cells with in KALTs, point to the central role of the RAS in the pathogenesis of KD (8). The RAS may influence the microenvironmental niche, through its interactions with VDR-mediated genomic mechanisms and the immune system, to regulate the ESC-like population within the KALTs, leading to the proliferation and accumulation of aberrant myofibroblasts and fibroblasts (Figure 9).

Vitamin D (VD) in the body exists in two forms: vitamin D₂ (VD₂, ergocalciferol) and vitamin D₃ (VD₃, cholecalciferol). VD₂ is gained from irradiation of plant sterol, ergosterol, and from dietary sources (206). Key determinants of VD₃ production include ultraviolent B intensity, which depends on latitude (204), and skin pigmentation which is determined by melanin pigment (207). Endogenous and exogenous VD are inactive and require two sequential hydroxylations to produce active VD (1,25-hydyoxyvitamin D₃) (208). The first hydroxylation step occurs in the liver, where 25-hydroxylation generates 25-OH-cholecalciferol (calcidiol). This then binds to VD binding protein and is released into the circulation to form the main circulating state of VD, and is therefore used as a marker of VD status (208). The second step is 1α-hydroxylation by 1α-hydroxylase, which is encoded by CYP27B1, and may be classified as renal-hydroxylation, occurring in the kidney, or extra-renal hydroxylation, occurring in other tissues. This converts 25-hydroxycholecalciferol (calcifediol) into the active form, 1,25-hydyoxyvitamin D₃ (calcitriol). This is largely responsible for the calcemic effects of VD. Extra-renal 1α-hydroxylation occurs in multiple organs and cell types – including kidney, brain, immune cells, lung, placenta and prostate (206). VD₂ and VD₃ will both be referred to as VD, due to their comparable biologic activity upon binding to VDR (209).

VD has its effect via VDR through both genomic and non-genomic actions (209). Its non-genomic actions include regulation of intestinal calcium and phosphorus absorption (210). For its genomic actions, VDR can change the expression of genes involved in cellular proliferation, stemness, and immunomodulation (206, 209). 1,25-hydyoxyvitamin D₃ binds to VDR, which then interacts with the retinoid X receptor which forms a heterodimer that binds to VD responsive elements near genes regulated by VD (208). This causes ligand-dependent induction or repression of gene transcription, alongside other recruited repressors and activators. The sheer quantity of target genes accounts for the broad pleiotropic actions of VDR (211). Given the presence of VDR in KLs, it is interesting to speculate its potential genomic actions on aberrant fibroblasts and myofibroblasts within KD, upon binding of VD (Figure 9).

Various extra-renal tissues express 1α-hydroxylase which produces 1,25-hydyoxyvitamin D₃ which acts locally in a tissue microenvironment via VDR. Notably, T and B lymphocytes produce the active form of VD. As mentioned, OCT4⁺ ESC-like cells within the KALTs of KLs express the VDR (19). It is interesting to speculate that active VD binds to VDR to exert genomic mechanisms on the ESC-like cells, and other cells expressing VDR in the tissue microenvironment to influence the local conditions to affect cell proliferation, stemness, and the chronic immune response seen in KD (8) (Figure 9). It is known that VD acts locally, regulating cell growth and proliferation within tissues such as the prostate and the placenta. Unlike renal 1α-hydroxylase, extra-renal 1α-hydroxylase is stimulated by cytokines, not parathyroid hormone (212).

DD affects the palmar fascia of the hand (213) with a predilection for those of Northern European descent, affecting almost 30% of the Norwegian population over 60 years (214). Fasciectomy is the first-line treatment for DD, which involves surgical removal of the fibrous cords and nodules, with a recurrence rate of up to 66% (215). More recently, non-operative treatment with Clostridium histolyticum collagenase injection has been used (216). The long-term outcomes and complications remain unknown, with repeated use being associated with neuromuscular and coagulation disorders (217, 218).

An ESC-like population expressing OCT4, NANOG, SOX2, pSTAT3 and SALL4 has been demonstrated on the endothelium of the microvessels in the cords and nodules of DD (21), in the location of a reported MSC population that expresses CD13 and CD29 (213). This MSC population may act as a reservoir for the aberrant myofibroblast population in DD (213) (Figure 10). Interestingly, dermatofasciectomy which involves excision of the overlying skin and surrounding tissues with full-thickness skin grafting, lowers the recurrence rate to 11.6% (215). This suggests the tissue surrounding the DD cords and nodules is involved in the disease process, potentially acting as a reservoir of the progenitor cells that give rise to aberrant myofibroblasts giving rise to DD (21, 217). Resident cells that differentiate into aberrant myofibroblasts in fibroproliferative conditions, have also been reported in other organs such as the liver and skin (8, 217).

The ESC-like population on the endothelium of the microvessels surrounding cords and nodules of DD expresses components of the RAS: PRR, ACE, AT₁R, AT₂R, with ACE and PRR also expressed on the pericytes (36). Stephen et al. (49) report AT₂R being more abundant than AT₁R in DD (218), which is interesting given the largely unknown role of AT₂R, which is expressed abundantly in fetal tissue compared to adult tissues. This suggests a potential therapeutic role of AT₂R antagonists and ACEIs for DD (219). The proteases cathepsins B, D and G, which constitute bypass loops of the RAS are expressed on the cords and nodules in DD with cathepsins B and D being expressed by the ESC-like cells on the endothelium of the microvessels (220). As the RAS is an important stem cell regulator (108), the expression of components of the RAS and cathepsin D on the ESC-like cells within DD, suggests this primitive population could be a potential therapeutic target by modulation of the RAS.

The ESC-like population on the microvessels has been proposed to be the source of the aberrant myofibroblasts in DD, via an MSC intermediate. This may occur by the primitive population acquiring an ESC-like phenotype, possibly under the influence of microenvironmental factors including the RAS, with a subsequent phenotypic shift giving rise to MSCs via endoMT, as proposed for KD (8).

Circulating CD34⁺ fibrocytes and MSCs from the bone marrow have also been proposed as another source of aberrant myofibroblasts (7) (Figure 9). Fibrocytes are fibroblast progenitor cells that originate from the bone marrow that migrate to distant sites where they participate in inflammation and fibrosis (219, 221). Circulating fibrocytes that exhibit a connective tissue myeloid cell phenotype are implicated in the pathogenesis of other fibroproliferative conditions. As progenitor cells, fibrocytes can give rise to cells of mesenchymal lineage, including myofibroblasts, under cues from the tissue microenvironment. Under the influence of growth factors, such as TGF-β and endothelin, fibrocytes gain the expression of α-smooth-muscle actin to turn into myofibroblast-like cells. The RAS cooperates with TGF-β to induce fibrosis via the AT₁R receptor in many tissue types (201).

There is a paucity of pre-clinical studies investigating the functional role of the RAS in DD with conflicting results, and there are no clinical studies investigating the effect of RASIs on DD (36, 219, 222, 223). There are a number pre-clinical and small clinical studies investigating the effect of RASIs in KD with mixed results (8). A pilot placebo-controlled trial investigating the effect of 5% topical losartan on 37 adults shows that the Vancouver scar scale scores reduce significantly in the losartan group with both KD and hypertrophic scars, with markedly reduced vascularity and improved pliability (222). Topical ACEI has been reported to improve KL in a case report of a post-burn patient (223), and in a study of two patients (224). Further investigations of the role of the RAS in KD and DD including functional studies and larger clinical trials are warranted to elucidate the effectiveness of RAS modulators in these fibroproliferative conditions.