KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses
Graphical abstract
Introduction
Cerebral cavernous malformations (CCMs), also known as cavernous angioma or cavernoma, are vascular anomalies typically found in the brain and spinal cord with a prevalence of 0.3%–0.5%. CCMs are mulberry-like, thin-walled sinusoidal capillaries lacking normal vessel structural components, including pericytes and astrocytes, and often surrounded by hemosiderin deposits and gliosis [[1], [2], [3]]. These vascular lesions can develop anywhere in the body, but signs and symptoms generally appear only when they occur in brain and spinal cord, where they account for 5–15% of all vascular malformations.
CCMs can occur as single or multiple lesions (even in the hundreds order), with size ranging from a few millimeters to a few centimeters. Despite the high prevalence of CCM lesions, approximately only 30% of affected people develop clinical symptoms, including recurrent headaches, neurological deficits, seizures, stroke, and intracerebral hemorrhage (ICH); however, the majority of CCM lesions remain clinically silent during most of the host's lifetime [3].
CCM is a disease of proven genetic origin that can arise sporadically or may be inherited as an autosomal dominant condition with incomplete penetrance and variable clinical expressivity [4]. The sporadic form (sCCM) accounts for up to 80% of cases, whereas the familial form (fCCM) accounts for at least 20% of cases. Genetic studies have identified three genes associated to CCMs: KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3), which account for about 50%, 20% and 10% of the cases, respectively. The remaining 20% are likely associated to undetected genetic alterations of CCM genes [5]. Many different CCM mutations have been identified, most of which typical of a single family. A different clinical penetrance between the CCM genes (60–88% for KRIT1, up to 100% for CCM2, and 63% for PDCD10) [6], and a large variability of severity of CCM lesions even among family members carrying the same germline mutation have been observed. However, distinct studies in animal models have clearly shown that mutations of CCM genes are not sufficient to cause CCM disease, suggesting that additional factors can contribute to CCM disease pathogenesis [5,7]. Consistently, DNA sequencing analysis of surgically-resected CCM lesions from autosomal dominant CCM patients have identified somatic mutations at very low frequencies, suggesting that the minority of cells in the mature CCM harbor these mutations [8,9].
In the central nervous system (CNS) the endothelium is part of complex units, called neurovascular units (NVU), where it is in close contact with other cell types (pericytes, astrocytes and neurons). All components of this unit interact with each other in a multidimensional process in which mediators released from multiple cells engage distinct signaling pathways and effector systems in a highly orchestrated manner and safeguard the integrity of the structure itself by regulating immune response, angiogenesis, vasculogenesis, oligodendrogenesis, neuroprotection and neuroplasticity [[10], [11], [12]].
To date, the effect of the KRIT1 loss on NVU is not known. However, recent observations outline the importance of microenvironment in the development of vascular lesions observed in fCCM. For instance, Louvi et al., demonstrated in an animal model that CCM3 neural deletion has cell nonautonomous effects resulting in the formation of multiple vascular lesions that closely resemble human cavernomas. Consistently, in a very recent paper Malinverno et al., showed that vascular lesions originate from clonal expansion of few CCM3 KO endothelial cells that attract sourrounding wild-type endothelial cells thus contributing to cavernoma growth [3,13,14].
Although the effective mechanisms through which loss of CCM proteins leads to vascular malformations remain to be comprehensively defined, in recent years it has been demonstrated that these proteins exert pleiotropic effects, related to their role in the regulation of multiple molecules and mechanisms involved in angiogenesis, cellular response to oxidative stress, inflammation, cell-cell and cell-matrix adhesion, and cytoskeleton dynamics [3,15].
Reactive oxygen species (ROS) are produced by the activity of a wide array of cellular enzymes, including NADPH oxidases (NOX), enzymes of the mitochondrial respiratory chain, xanthine oxidases, cytochrome p450 monooxygenases, lipoxygenases and cyclooxygenases, which can be induced by a variety of endogenous and exogenous chemical and physical stimuli [16]. The NOX family of enzymes produces ROS as their sole function, and are becoming recognized as key modulators of signal transduction pathways with a physiological role under acute stress and a pathological role after excessive activation under chronic stress. ROS produced by NOX proteins are now recognized to play essential roles in the regulation of cytoskeletal remodeling, gene expression, proliferation, differentiation, migration, and cell death. The NOX isoforms (NOX1–5, and DUOX1/2) differ in their regulation, tissue and subcellular localization and even ROS products [17,18]. NOX1, NOX2, NOX4, and NOX5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. While NOX1/NOX2 promote the development of endothelial dysfunction, hypertension, and inflammation, NOX4 may play a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental [19]. Recently NOX1 has been involved in several brain diseases [20], and has been also described to play a role in cancer by inducing tumor progression and angiogenesis through the regulation of vascular endothelial growth factor (VEGF) expression [[21], [22], [23]].
Previously, we demonstrated that KRIT1 loss affects the intracellular redox homeostasis and results in increased ROS production through distinct mechanisms, including Forkhead box protein O1 (FoxO1) and superoxide dismutase (SOD) downregulation, NOX4 upregulation, and abnormal antioxidant responses, suggesting a novel pathogenetic mechanism whereby CCM disease may result from impaired endothelial cell defenses to microenvironmental oxidative stress events [4,16,[24], [25], [26], [27], [28], [29], [30]].
Herein, we show that KRIT1 loss-of-function in fibroblasts induces the upregulation of NOX1, which in turn can trigger a paracrine proangiogenic response in wild type endothelial cells through increased production and release of angiogenic growth factors, suggesting a novel important role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM disease pathogenesis, and pointing to NOX1 as a major regulator of these effects and as a new potential therapeutic target.
Section snippets
Cell culture
Wild-type (K+/+) and KRIT1 knock-out (K−/−) mouse embryonic fibroblast (MEF) cell lines were established from KRIT1+/+ and KRIT1−/− E8.5 mouse embryos, respectively [28]. KRIT1−/− MEFs re-expressing KRIT1 (K9/6) were obtained as previously reported [28]. Cells were cultured at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), with 4500 mg/l glucose and 100 U/ml penicillin/streptomycin (Euroclone, Milan, Italy).
Human Umbilical Vein
Conditioned medium from KRIT1−/− fibroblasts induces pro-angiogenic responses in wild type human endothelial cells
Accumulated evidences demonstrate that KRIT1 protein plays an important role in modulating different molecular pathways involved in the angiogenic process, whereas KRIT1 loss induces alteration of endothelial cell-cell and cell-extracellular matrix (ECM) adhesion, and enhanced vascular permeability [3,36,37]. However, recent evidence in conditional knockout (cKO) animal models has clearly shown that loss-of-function mutations in CCM genes alone are not sufficient for the development of CCM
Discussion
CCM lesions are typically found in the CNS and are characterized by dilated and leaky capillaries that are devoid of normal vessel structural components.
Several lines of evidence show that the KRIT1 protein is involved in different physiological aspects of endothelial biology, including vascular development, modulation of different redox-sensitive signaling pathways, and maintenance of endothelial barrier homeostasis, as well as that the absence of KRIT1 in endothelial cells induces an
Conclusions
Our findings provide novel insights into CCM pathogenesis, and suggest novel promising therapeutic options for CCM prevention and treatment.
Currently, the only therapy available for CCM is surgical excision or radiological destruction of the lesions. While several compounds are being investigated in preclinical studies, only a few agents have reached clinical testing and to date CCM has no pharmacological options [52]. Several studies have been conducted to identify new CCM-related cellular
Funding
This work was supported by the Telethon Foundation (grant GGP15219) to SFR and LT and MIUR (Progetto Dipartimento di Eccellenza 2018-2022) to LT and FF.
Acknowledgements
The authors are grateful to the Italian Research Network for Cerebral Cavernous Malformation (CCM Italia, http://www.ccmitalia.unito.it), and the Associazione Italiana Angiomi Cavernosi (AIAC, http://www.ccmitalia.unito.it/aiac) for fundamental collaboration and support.
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These authors contributed equally to this work.
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CCM Italia research network (www.ccmitalia.unito.it).