Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Podocyte Injury in Diabetic Kidney Disease in Mouse Models Involves TRPC6-mediated Calpain Activation Impairing Autophagy.

SIGNIFICANCE STATEMENT: Autophagy protects podocytes from injury in diabetic kidney disease (DKD). Restoring glomerular autophagy is a promising approach to limit DKD. This study demonstrates a novel regulatory mechanism of autophagy that blocks this critical protection of the glomerular filtration barrier. We demonstrated that TRPC6 induced in podocytes in mouse models of diabetes mediates calpain activation, thereby impairing podocyte autophagy, causing injury and accelerating DKD. Furthermore, this study provides proof of principle for druggable targets for DKD because restoration of podocyte autophagy by calpain inhibitors effectively limits glomerular destruction. BACKGROUND: Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury. The regulation of podocyte autophagy is unique because it minimally uses the mTOR and AMPK pathways. Thus, the molecular mechanisms underlying the impaired autophagy in podocytes in diabetic kidney disease remain largely elusive. METHODS: This study investigated how the calcium channel TRPC6 and the cysteine protease calpains deleteriously affect podocyte autophagy in diabetic kidney disease in mice. We demonstrated that TRPC6 knockdown in podocytes increased the autophagic flux because of decreased cysteine protease calpain activity. Diabetic kidney disease was induced in vivo using streptozotocin with unilateral nephrectomy and the BTBR ob/ob mouse models. RESULTS: Diabetes increased TRPC6 expression in podocytes in vivo with decreased podocyte autophagic flux. Transgenic overexpression of the endogenous calpain inhibitor calpastatin, as well as pharmacologic inhibition of calpain activity, normalized podocyte autophagic flux, reduced nephrin loss, and prevented the development of albuminuria in diabetic mice. In kidney biopsies from patients with diabetes, we further confirmed that TRPC6 overexpression in podocytes correlates with decreased calpastatin expression, autophagy blockade, and podocyte injury. CONCLUSIONS: Overall, we discovered a new mechanism that connects TRPC6 and calpain activity to impaired podocyte autophagy, increased podocyte injury, and development of proteinuria in the context of diabetic kidney disease. Therefore, targeting TRPC6 and/or calpain to restore podocyte autophagy might be a promising therapeutic strategy for diabetic kidney disease.

From bench to bedside: lessons learned from translational podocyte research.

Polat et al. report that mice with a podocyte-specific expression of a constitutively active Rac1 form displayed similar injury and albuminuria, regardless of transient receptor potential canonical 5 activity. This article confirms the pathogenic role of deregulated Rac1 and challenges models involving the role of transient receptor potential canonical 5 in podocytes. We learned from this study and propose a roadmap for this controversial field to help new drug candidates succeed in clinical trials and safely reach patients.

FIBER-ML, an Open-Source Supervised Machine Learning Tool for Quantification of Fibrosis in Tissue Sections.

Pathologic fibrosis is a major hallmark of tissue insult in many chronic diseases. Although the amount of fibrosis is recognized as a direct indicator of the extent of disease, there is no consentaneous method for its quantification in tissue sections. This study tested FIBER-ML, a semi-automated, open-source freeware that uses a machine-learning approach to quantify fibrosis automatically after a short user-controlled learning phase. Fibrosis was quantified in sirius red-stained tissue sections from two fibrogenic animal models: acute stress-induced cardiomyopathy in rats (Takotsubo syndrome-like) and HIV-induced nephropathy in mice (chronic kidney disease). The quantitative results of FIBER-ML software version 1.0 were compared with those of ImageJ in Takotsubo syndrome, and with those of inForm in chronic kidney disease. Intra- and inter-operator and inter-software correlation and agreement were assessed. All correlations were excellent (>0.95) in both data sets. The values of discriminatory power between the pathologic and healthy groups were <10-3 for data on Takotsubo syndrome and <10-4 for data on chronic kidney disease. Intra-operator agreement, assessed by intra-class coefficient correlation, was good (>0.8), while inter-operator and inter-software agreement ranged from moderate to good (>0.7). FIBER-ML performed in a fast and user-friendly manner, with reproducible and consistent quantification of fibrosis in tissue sections. It offers an open-source alternative to currently used software, including quality control and file management.

ScoMorphoFISH: A deep learning enabled toolbox for single-cell single-mRNA quantification and correlative (ultra-)morphometry.

Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single-cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to obtain spatial single-cell single-mRNA expression data from routinely generated kidney biopsies. Deep learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single-cell expression data were assigned. Furthermore, we show correlative and spatial single-cell expression quantification with super-resolved podocyte foot process morphometry. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single-cell level with single-mRNA resolution. Using this method, we demonstrate that ACE2 can be locally upregulated in podocytes upon injury. In a patient suffering from COVID-19-associated collapsing FSGS, ACE2 expression levels were correlated with intracellular SARS-CoV-2 abundance. As this method performs well with standard formalin-fixed paraffin-embedded samples and we provide pretrained deep learning networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings.

Should we consider calcimimetics as a therapeutic option for nephrotic syndrome?

Calcimimetics allosterically increase the calcium ion sensitivity of the calcium-sensing receptor (CaSR). Using a CaSR knockdown in podocytes and a podocyte-specific CaSR knockout in mice, Mühlig et al. uncovered a stabilizing role for actin cytoskeleton and cell adhesion. Short-term alleviation of albuminuria and proteinuria was observed in 4 children treated with cinacalcet. Here we discuss the potential mechanisms whereby CaSR displays a favorable effect in podocytes and the context in which calcimimetics may alleviate nephrotic syndrome.

Calpastatin prevents Angiotensin II-mediated podocyte injury through maintenance of autophagy.

The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.

Podocyte healthy self-eating boosted by a spermidine meal?

The mechanisms sustaining a high level of autophagy in podocytes are not well delineated. Seminal studies had unraveled that the polyamine pathway is involved in the regulation of aging and autophagy. Polyamines (e.g., spermine, spermidine, and putrescine) are ubiquitous molecules essential for the physiological processes, including cell growth, development, and differentiation. Liang et al. examined the role of ornithine decarboxylase, and spermidine synthase, and demonstrated that endogenous spermidine is required to maintain intact podocyte autophagy.

Deletion of the myeloid endothelin-B receptor confers long-term protection from angiotensin II-mediated kidney, eye and vessel injury.

The endothelin system may be an important player in hypertensive end-organ injury as endothelin-1 increases blood pressure and is pro-inflammatory. The immune system is emerging as an important regulator of blood pressure and we have shown that the early hypertensive response to angiotensin-II infusion was amplified in mice deficient of myeloid endothelin-B (ETB) receptors (LysM-CreEdnrblox/lox). Hypothesizing that these mice would display enhanced organ injury, we gave angiotensin-II to LysM-CreEdnrblox/lox and littermate controls (Ednrblox/lox) for six weeks. Unexpectedly, LysM-CreEdnrblox/lox mice were significantly protected from organ injury, with less proteinuria, glomerulosclerosis and inflammation of the kidney compared to controls. In the eye, LysM-CreEdnrblox/lox mice had fewer retinal hemorrhages, less microglial activation and less vessel rarefaction. Cardiac remodeling and dysfunction were similar in both groups at week six but LysM-CreEdnrblox/lox mice had better endothelial function. Although blood pressure was initially higher in LysM-CreEdnrblox/lox mice, this was not sustained. A natriuretic switch at about two weeks, due to enhanced ETB signaling in the kidney, induced a hypertensive reversal. By week six, blood pressure was lower in LysM-CreEdnrblox/lox mice than in controls. At six weeks, macrophages from LysM-CreEdnrblox/lox mice were more anti-inflammatory and had greater phagocytic ability compared to the macrophages of Ednrblox/lox mice. Thus, myeloid cell ETB receptor signaling drives this injury both through amplifying hypertension and by inflammatory polarization of macrophages.

Parietal epithelial cells role in repair versus scarring after glomerular injury.

PURPOSE OF REVIEW: The recent years have been marked by the publication of several articles highlighting the pathophysiological role of glomerular parietal epithelial cells (PEC) and refining their phenotypic heterogeneity. RECENT FINDINGS: The present review synthetizes recent findings on (i) the potential regenerative role of PEC in glomerular diseases, and (ii) the mechanisms and signaling of leading to PEC pathogenic involvement in crescentic glomerulonephritis (CGN) and focal segmental glomerulosclerosis (FSGS). SUMMARY: The debate is still open regarding the podocyte regenerative properties of PEC in glomerular disease, whereas the pathogenic involvement of PEC activation in glomerular disease is increasingly admitted. Recent highlights on the podocyte regenerative role of PEC, on one hand, and on their pathological function, on the other hand, for sure will feed the debate in the kidney community for the next years. Nevertheless, from a therapeutic perspective, the two options, boosting cellular regeneration and blocking PECs pathogenicity, should not be seen as antagonistic but, rather, complementary.

A novel role for myeloid endothelin-B receptors in hypertension.

AIMS: Hypertension is common. Recent data suggest that macrophages (Mφ) contribute to, and protect from, hypertension. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor with additional pro-inflammatory properties. We investigated the role of the ET system in experimental and clinical hypertension by modifying Mφ number and phenotype. METHODS AND RESULTS: In vitro, Mφ ET receptor function was explored using pharmacological, gene silencing, and knockout approaches. Using the CD11b-DTR mouse and novel mice with myeloid cell-specific endothelin-B (ETB) receptor deficiency (LysMETB-/-), we explored the effects of modifying Mφ number and phenotype on the hypertensive effects of ET-1, angiotensin II (ANG II), a model that is ET-1 dependent, and salt. In patients with small vessel vasculitis, the impacts of Mφ depleting and non-depleting therapies on blood pressure (BP) and endothelial function were examined. Mouse and human Mφ expressed both endothelin-A and ETB receptors and displayed chemokinesis to ET-1. However, stimulation of Mφ with exogenous ET-1 did not polarize Mφ phenotype. Interestingly, both mouse and human Mφ cleared ET-1 through ETB receptor mediated, and dynamin-dependent, endocytosis. Mφ depletion resulted in an augmented chronic hypertensive response to both ET-1 and salt. LysMETB-/- mice displayed an exaggerated hypertensive response to both ET-1 and ANG II. Finally, in patients who received Mφ depleting immunotherapy BP was higher and endothelial function worse than in those receiving non-depleting therapies. CONCLUSION: Mφ and ET-1 may play an important role in BP control and potentially have a critical role as a therapeutic target in hypertension.

Cardiac arrest in takotsubo syndrome: results from the InterTAK Registry.

AIMS: We aimed to evaluate the frequency, clinical features, and prognostic implications of cardiac arrest (CA) in takotsubo syndrome (TTS). METHODS AND RESULTS: We reviewed the records of patients with CA and known heart rhythm from the International Takotsubo Registry. The main outcomes were 60-day and 5-year mortality. In addition, predictors of mortality and predictors of CA during the acute TTS phase were assessed. Of 2098 patients, 103 patients with CA and known heart rhythm during CA were included. Compared with patients without CA, CA patients were more likely to be younger, male, and have apical TTS, atrial fibrillation (AF), neurologic comorbidities, physical triggers, and longer corrected QT-interval and lower left ventricular ejection fraction on admission. In all, 57.1% of patients with CA at admission had ventricular fibrillation/tachycardia, while 73.7% of patients with CA in the acute phase had asystole/pulseless electrical activity. Patients with CA showed higher 60-day (40.3% vs. 4.0%, P < 0.001) and 5-year mortality (68.9% vs. 16.7%, P < 0.001) than patients without CA. T-wave inversion and intracranial haemorrhage were independently associated with higher 60-day mortality after CA, whereas female gender was associated with lower 60-day mortality. In the acute phase, CA occurred less frequently in females and more frequently in patients with AF, ST-segment elevation, and higher C-reactive protein on admission. CONCLUSIONS: Cardiac arrest is relatively frequent in TTS and is associated with higher short- and long-term mortality. Clinical and electrocardiographic parameters independently predicted mortality after CA.

Endothelial Epas1 Deficiency Is Sufficient To Promote Parietal Epithelial Cell Activation and FSGS in Experimental Hypertension.

FSGS, the most common primary glomerular disorder causing ESRD, is a complex disease that is only partially understood. Progressive sclerosis is a hallmark of FSGS, and genetic tracing studies have shown that parietal epithelial cells participate in the formation of sclerotic lesions. The loss of podocytes triggers a focal activation of parietal epithelial cells, which subsequently form cellular adhesions with the capillary tuft. However, in the absence of intrinsic podocyte alterations, the origin of the pathogenic signal that triggers parietal epithelial cell recruitment remains elusive. In this study, investigation of the role of the endothelial PAS domain-containing protein 1 (EPAS1), a regulatory α subunit of the hypoxia-inducible factor complex, during angiotensin II-induced hypertensive nephropathy provided novel insights into FSGS pathogenesis in the absence of a primary podocyte abnormality. We infused angiotensin II into endothelial-selective Epas1 knockout mice and their littermate controls. Although the groups presented with identical high BP, endothelial-specific Epas1 gene deletion accentuated albuminuria with severe podocyte lesions and recruitment of pathogenic parietal glomerular epithelial cells. These lesions and dysfunction of the glomerular filtration barrier were associated with FSGS in endothelial Epas1-deficient mice only. These results indicate that endothelial EPAS1 has a global protective role during glomerular hypertensive injuries without influencing the hypertensive effect of angiotensin II. Furthermore, these findings provide proof of principle that endothelial-derived signaling can trigger FSGS and illustrate the potential importance of the EPAS1 endothelial transcription factor in secondary FSGS.

The endothelin B receptor plays a crucial role in the adhesion of neutrophils to the endothelium in sickle cell disease.

Although the primary origin of sickle cell disease is a hemoglobin disorder, many types of cells contribute considerably to the pathophysiology of the disease. The adhesion of neutrophils to activated endothelium is critical in the pathophysiology of sickle cell disease and targeting neutrophils and their interactions with endothelium represents an important opportunity for the development of new therapeutics. We focused on endothelin-1, a mediator involved in neutrophil activation and recruitment in tissues, and investigated the involvement of the endothelin receptors in the interaction of neutrophils with endothelial cells. We used fluorescence intravital microscopy analyses of the microcirculation in sickle mice and quantitative microfluidic fluorescence microscopy of human blood. Both experiments on the mouse model and patients indicate that blocking endothelin receptors, particularly ETB receptor, strongly influences neutrophil recruitment under inflammatory conditions in sickle cell disease. We show that human neutrophils have functional ETB receptors with calcium signaling capability, leading to increased adhesion to the endothelium through effects on both endothelial cells and neutrophils. Intact ETB function was found to be required for tumor necrosis factor α-dependent upregulation of CD11b on neutrophils. Furthermore, we confirmed that human neutrophils synthesize endothelin-1, which may be involved in autocrine and paracrine pathophysiological actions. Thus, the endothelin-ETB axis should be considered as a cytokine-like potent pro-inflammatory pathway in sickle cell disease. Blockade of endothelin receptors, including ETB, may provide major benefits for preventing or treating vaso-occlusive crises in sickle cell patients.

Endothelin-1 Induces Proteinuria by Heparanase-Mediated Disruption of the Glomerular Glycocalyx.

Diabetic nephropathy (DN) is the leading cause of CKD in the Western world. Endothelin receptor antagonists have emerged as a novel treatment for DN, but the mechanisms underlying the protective effect remain unknown. We previously showed that both heparanase and endothelin-1 are essential for the development of DN. Here, we further investigated the role of these proteins in DN, and demonstrated that endothelin-1 activates podocytes to release heparanase. Furthermore, conditioned podocyte culture medium increased glomerular transendothelial albumin passage in a heparanase-dependent manner. In mice, podocyte-specific knockout of the endothelin receptor prevented the diabetes-induced increase in glomerular heparanase expression, consequent reduction in heparan sulfate expression and endothelial glycocalyx thickness, and development of proteinuria observed in wild-type counterparts. Our data suggest that in diabetes, endothelin-1 signaling, as occurs in endothelial activation, induces heparanase expression in the podocyte, damage to the glycocalyx, proteinuria, and renal failure. Thus, prevention of these effects may constitute the mechanism of action of endothelin receptor blockers in DN.

Nuclear Factor Erythroid 2-Related Factor 2 Drives Podocyte-Specific Expression of Peroxisome Proliferator-Activated Receptor γ Essential for Resistance to Crescentic GN.

Necrotizing and crescentic rapidly progressive GN (RPGN) is a life-threatening syndrome characterized by a rapid loss of renal function. Evidence suggests that podocyte expression of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) may prevent podocyte injury, but the function of glomerular PPARγ in acute, severe inflammatory GN is unknown. Here, we observed marked loss of PPARγ abundance and transcriptional activity in glomerular podocytes in experimental RPGN. Blunted expression of PPARγ in podocyte nuclei was also found in kidneys from patients diagnosed with crescentic GN. Podocyte-specific Pparγ gene targeting accentuated glomerular damage, with increased urinary loss of albumin and severe kidney failure. Furthermore, a PPARγ gain-of-function approach achieved by systemic administration of thiazolidinedione (TZD) failed to prevent severe RPGN in mice with podocyte-specific Pparγ gene deficiency. In nuclear factor erythroid 2-related factor 2 (NRF2)-deficient mice, loss of podocyte PPARγ was observed at baseline. NRF2 deficiency markedly aggravated the course of RPGN, an effect that was partially prevented by TZD administration. Furthermore, delayed administration of TZD, initiated after the onset of RPGN, still alleviated the severity of experimental RPGN. These findings establish a requirement for the NRF2-PPARγ cascade in podocytes, and we suggest that these transcription factors have a role in augmenting the tolerance of glomeruli to severe immune-complex mediated injury. The NRF2-PPARγ pathway may be a therapeutic target for RPGN.

Autophagy in kidney disease and aging: lessons from rodent models.

Autophagy is a highly regulated lysosomal protein degradation pathway that removes protein aggregates and damaged or excess organelles to maintain intracellular homeostasis and cell integrity. Dysregulation of autophagy is involved in the pathogenesis of a variety of metabolic and age-related diseases. Growing evidence suggests that autophagy is implicated in cell injury during renal diseases, both in the tubulointerstitial compartment and in glomeruli. Nevertheless, the impact of autophagy on renal disease progression and aging is still not fully understood. This review summarizes the recent advances in understanding the role of autophagy for kidney disease and aging.

Direct action of endothelin-1 on podocytes promotes diabetic glomerulosclerosis.

The endothelin system has emerged as a novel target for the treatment of diabetic nephropathy. Endothelin-1 promotes mesangial cell proliferation and sclerosis. However, no direct pathogenic effect of endothelin-1 on podocytes has been shown in vivo and endothelin-1 signaling in podocytes has not been investigated. This study investigated endothelin effects in podocytes during experimental diabetic nephropathy. Stimulation of primary mouse podocytes with endothelin-1 elicited rapid calcium transients mediated by endothelin type A receptors (ETARs) and endothelin type B receptors (ETBRs). We then generated mice with a podocyte-specific double deletion of ETAR and ETBR (NPHS2-Cre×Ednra(lox/lox)×Ednrb(lox/lox) [Pod-ETRKO]). In vitro, treatment with endothelin-1 increased total ß-catenin and phospho-NF-κB expression in wild-type glomeruli, but this effect was attenuated in Pod-ETRKO glomeruli. After streptozotocin injection to induce diabetes, wild-type mice developed mild diabetic nephropathy with microalbuminuria, mesangial matrix expansion, glomerular basement membrane thickening, and podocyte loss, whereas Pod-ETRKO mice presented less albuminuria and were completely protected from glomerulosclerosis and podocyte loss, even when uninephrectomized. Moreover, glomeruli from normal and diabetic Pod-ETRKO mice expressed substantially less total ß-catenin and phospho-NF-κB compared with glomeruli from counterpart wild-type mice. This evidence suggests that endothelin-1 drives development of glomerulosclerosis and podocyte loss through direct activation of endothelin receptors and NF-κB and ß-catenin pathways in podocytes. Notably, both the expression and function of the ETBR subtype were found to be important. Furthermore, these results indicate that activation of the endothelin-1 pathways selectively in podocytes mediates pathophysiologic crosstalk that influences mesangial architecture and sclerosis.

BMP9 is a key player in endothelial identity and its loss is sufficient to induce arteriovenous malformations.

AIMS: BMP9 is a high affinity ligand of ALK1 and endoglin receptors that are mutated in the rare genetic vascular disorder hereditary hemorrhagic telangiectasia (HHT). We have previously shown that loss of Bmp9 in the 129/Ola genetic background leads to spontaneous liver fibrosis via capillarization of liver sinusoidal endothelial cells (LSEC) and kidney lesions. We aimed to decipher the molecular mechanisms downstream of BMP9 to better characterize its role in vascular homeostasis in different organs. METHODS AND RESULTS: For this, we performed an RNA-seq analysis on LSEC from adult WT and Bmp9-KO mice and identified over 2000 differentially expressed genes. Gene ontology analysis showed that Bmp9 deletion led to a decrease in BMP and Notch signalling, but also LSEC capillary identity while increasing their cell cycle. The gene ontology term 'glomerulus development' was also negatively enriched in Bmp9-KO mice vs. WT supporting a role for BMP9 in kidney vascularization. Through different imaging approaches (electron microscopy, immunostainings), we found that loss of Bmp9 led to vascular enlargement of the glomeruli capillaries associated with alteration of podocytes. Importantly, we also showed for the first time that the loss of Bmp9 led to spontaneous arteriovenous malformations (AVMs) in the liver, gastrointestinal tract, and uterus. CONCLUSION: Altogether, these results demonstrate that BMP9 plays an important role in vascular quiescence both locally in the liver by regulating endothelial capillary differentiation markers and cell cycle but also at distance in many organs via its presence in the circulation. It also reveals that loss of Bmp9 is sufficient to induce spontaneous AVMs, supporting a key role for BMP9 in the pathogenesis of HHT.