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Zhang et al. Vessel Plus 2021;5:48 https://dx.doi.org/10.20517/2574-1209.2021.64 Page 5 of 14

increased PRG levels during early pregnancy [116,117] have been indicated to enhance the progression of
[119]
[118]
lesions , possibly through the induction of structural changes within vessels . Increased risk for acute
CCM bleedings [101,103,104,113,115,120] or formation of a de novo CCM lesion [113,120,121] have also been reported during
pregnancy. Collectively, these findings reinforce the idea that there is gender- and sex hormone-associated
differences in hemorrhagic stroke pathophysiology and suggest that PRG-mediated signaling should be
further investigated. Interestingly, nPR(+) endothelial cells (ECs) can only be found in the veins and
lymphatics of the uterus and ovaries, where human umbilical vein endothelial cells (HUVECs) are
derived . Additionally, the MEKK3-KLF-ADAMTS signaling pathway has been implicated in CSC-
[122]
mediated angiogenic activities . KLFs are largely expressed in reproductive tissues and have been
[123]
[124]
implicated as co-regulators and integrators of progesterone/progesterone receptor transactivity . There
are some associations between progesterone and ADAMTS-1 demonstrating ADAMTS as a transcriptional
target of progesterone actions mainly in the ovaries of nPR-KO mice . Additionally, in Xenopus laevis eggs
[125]
and embryos, it was demonstrated that progesterone can stimulate JNK activation through both MEK/p42
MAPK-dependent and -independent pathways, and the addition of progesterone induced synthesis of
MAPKKK c-Mos, leading to the activation of the MEK1-ERK-RSK cascade . However, there is currently
[126]
no available literature systemically demonstrating the effects of progesterone on the MEKK3-KLF-
ADAMTS signaling pathway. The vast majority of vascular ECs derived from other tissues are nPR(-) and
mPRs(+), where only non-classic actions of PRG have been reported . When we used combined steroids
[82]
(PRG + MIF) to treat four nPRs(-) microvascular ECs and nPRs(+) HUVECs, again, our data support a
common regulatory mechanism underlying the inhibitory effects of PRG/MIF on CSC, independent of
nPRs. In addition, the sex hormone inhibition of CCM1/3 protein expression in ECs is more dramatic than
in non-endothelial-derived cell lines [51,127] , reaffirming that steroid hormones have much stronger actions on
the stability of CSC through mPRs in ECs.

PRG ACTIONS INCREASED PERMEABILITY OF EC MONOLAYER AND COMPROMISED
BBB INTEGRITY
The haploinsufficiency of CCM proteins in microvascular ECs is an essential step in the pathogenesis of
CCM lesions, as demonstrated by in vivo studies with zebrafish [128,129] and Ccms mice models [130-132] , but it is
insufficient to form hemorrhagic CCMs. Although the “two-hit” model, which creates a null condition in
the lesion, can be used to explain familial CCM cases, it fails to account for sporadic forms of CCM, which
make up 80% of all CCM cases . Additional studies have demonstrated that haploinsufficiency condition
[133]
of CCMs are insufficient in initiating hemorrhagic events of CCM lesions . Since the “two-hit” model
[134]
alone cannot explain CCM ruptures, there must be a molecular “trigger” that initiates the hemorrhagic
events of CCM lesions. Therefore, we performed both in vivo and in vitro permeability assays
demonstrating significantly increased blood-brain barrier (BBB) permeability among all Ccms (1, 2, 3)
mutants only in the hormone treatment groups, compared to WT and/or untreated Ccms (1, 2, 3) mutants
mice, which was further supported by in vitro permeability assays showing increased permeability of
different EC lines under steroid actions, compared to vehicle controls [51,127] . This concordant BBB leakage
among all Ccms mutant mice was not seen in other treatment groups, nor that of other tested organs,
indicating that chronic steroid actions specifically increase BBB permeability, and it is the primary
mechanism underlying CCM lesion formation. Therefore, we concluded that BBB integrity among
[51]
individuals with CCMs deficiency is particularly susceptible to chronic and elevated sex steroid actions .
PARADIGM SHIFT FOR HEMORRHAGIC EVENTS IN CCMS OPENS UP NEW AVENUES OF
RESEARCH
Hemorrhage is often rooted in defective endothelial cell junctions, and microvessel rupture is a result of
compromised BBB integrity . Currently, two major theories for the induction of hemorrhagic CCMs are
[135]