Cardiovascular Insights from a Premature Aging Syndrome: A Translational Story

Abstract

Good morning. I am delighted to join you this morning. Today I'd like to tell you an interesting translational research story whereby a rare disease can be an example for how scientific advances can lead to acceleration of new therapies. This story will include some genetic sleuthing, a creative interdisciplinary team, a foundation of protein biochemistry, cutting-edge cell biology, creation of a mouse model, designer drug development, some good luck along the way, kids enrolling in a clinical trial from around the world, and finally, unexpected consequences for a condition that affects all of us.

GENETICS, MOLECULAR BIOLOGY, AND BIOCHEMISTRY

Hutchinson-Gilford Progeria Syndrome (HGPS) was first described by Jonathan Hutchinson in 1886 (1). It is a rare genetic disorder, affecting approximately one in 4 million live births. The clinical features include premature aging, growth retardation, alopecia, diminished subcutaneous fat, bone reabsorption and arthritis, and premature death in the early teens due to heart attack and/or stroke.

The genetic cause of HGPS was a mystery during the 191^th and 20^th centuries. Dr. Francis Collins and his laboratory discovered the genetic etiology in 2003; namely, a point mutation in exon 11 of the lamin A or LMNA gene (2). A spontaneous mutation in position 608 within exon 11 changes a GGC sequence to a GGT sequence (G608G). Both codons encode glycine, so there is no change in the amino acid sequence. Rather, a cryptic splice site is activated leading to an internal deletion of 150 nucleotides, or 50 amino acids, in the 3'-terminus of exon 11 during RNA processing. A shortened protein, known as progerin, is produced during translation, rather than the normal protein product, prelamin A.

After translation, prelamin A undergoes biochemical modification to mature lamin A. The first step, farnesylation, involves the addition of a 15-carbon isoprenoid lipid moiety to the C-terminal CAAX motif, which targets the prelamin A precursor to the nuclear envelope. The protein then undergoes cleavage of the terminal 3 amino acids followed by carboxymethylation. The enzyme Zmpste24 cleaves the terminal 15 amino acids, including the farnesyl group, forming the mature 72-kDa lamin A that inserts into the nuclear lamina. The mutant lamin A protein, or progerin, has a 50-amino-acid internal deletion that removes the cleavage site for Zmpste24, and remains permanently farnesylated and permanently anchored to the nuclear envelope.

Cell biology studies have been performed using dermal fibroblasts from HGPS individuals and their parents without HGPS. Progerin anchoring to the nuclear envelope disrupts the architecture of the nuclear lamina, leading to the characteristic morphological features of blebbing and pynotic nuclei. Other functions of the nucleus are also altered, including gene transcription and chromatin remodeling. Genomic instability and mechanical disruption in HGPS is reviewed by our group in 2007 (3).

CARDIOVASCULAR PHENOTYPE

The pathophysiology of cardiovascular disease in HGPS has been poorly understood due in large part to the lack of autopsy or biopsy materials reported in the literature. A comprehensive autopsy report was provided by Stehbens et al (4) who examined the blood vessels in a 22-year-old woman who had clinical features of HGPS (but no genetic diagnosis). Dr. Stehbens described a dramatic loss of vascular smooth muscle cells in the medial layer of all arteries and veins, acellular intimal thickening, loss of endothelial cells, and spontaneous breaks in elastic structures. Extracellular matrix, predominantly collagen, replaced vascular smooth muscle and endothelial cells, leading to a chronic fibrosis. We then hypothesized from the work of Stehbens et al (4) that this pathological vascular remodeling — loss of contractile vascular smooth muscle cells and replacement by a fibrous sheath — leads to reduced arterial elasticity and susceptibility to mechanical and hemodynamic stress.

We recently examined the coronary arteries in two autopsy cases of children with genetic proof of HGPS (G608G mutation) (5). These two children died of myocardial infarction and were found to have a diffuse loss of vascular smooth muscle and endothelial cells throughout their coronary arteries with replacement by fibrosis and adventitial thickening. Progerin was detected in the HGPS arteries and in the coronary arteries of non-HGPS individuals with coronary artery disease, suggesting that progerin may play a role in cardiovascular aging in the normal population.

MOUSE MODEL OF HGPS

To further investigate the natural history of HGPS and to facilitate development of a therapy, we created a mouse model of HGPS that recreates the human cardiovascular phenotype (6). A BAC clone expressing human progerin (G608G mutation in LMNA) was inserted into mouse DNA creating a transgenic mouse, and both single and double copy mice were bred. We then conducted a natural history study for which we examined vascular pathology at various intervals extending to 12 months. We observed a progressive loss of medial vascular smooth muscle cells, spontaneous breaks in elastin, and replacement by collagen and proteoglycans by 5 months of age in the aorta and carotid arteries. By 12 months, these arteries were devoid of smooth muscle and endothelial cells with extensive collagen and proteoglycan deposition extending into the adventitia, creating a fibrous sheath around a degenerative medial. We further tested the functional consequences of these profound anatomical changes by infusing the vasodilator, sodium nitroprusside, into the tail vein while measuring blood pressure in the carotid artery. The littermate control mice showed a normal decrease in blood pressure when challenged with sodium nitroprusside; the HGPS mice exhibited hypotension at baseline, a marked blunting of blood pressure in response to sodium nitroprusside, and minimal recovery over time, reflecting an absence of medial smooth muscle cells that vasocontrict or vasodilate.

DEVELOPING A THERAPY

The toxic cellular effects of the farnesylated progerin protein led to the hypothesis that a reduction in progerin, even if incomplete, might improve cellular function. Farnesyltransferase inhibitors (FTIs) are orally available and in phase III investigation for cancers, suggesting a possible therapeutic option for HGPS. Accordingly, we conducted in vitro studies in HGPS dermal fibroblasts with FTIs. Dose-dependent treatment of HGPS fibroblasts with FTIs restored the normal nuclear shape (7). We then proceeded to two clinical trials of FTIs in our mouse model of HGPS (8). The first trial examined whether the onset of vascular disease could be prevented by the early treatment of the HGPS mouse with a FTI. Indeed, 9 to 12 months of oral FTI treatment in HGPS mice starting at 1 month of age prevented vascular smooth muscle and endothelial cell loss. The second trial examined whether FTI treatment initiated in HGPS mice at a later age (similar to starting treatment in older HGPS children) would prevent the late progression of cardiovascular disease; indeed, at the higher doses of FTIs, vascular cell loss was prevented and vascular architecture was preserved. These observations provided encouraging evidence and provided the basis for the current clinical trial of FTIs in HGPS (9).

PROGERIN AND NORMAL AGING

An interesting finding is that progerin is produced at low levels in normal tissues and increases with age (10). We previously reported that progerin is present in the coronary arteries of individuals with coronary artery disease as well as in low levels in normal coronary arteries (5). Progerin has also been detected in human skin where it is a biomarker of cellular aging (11). We recently examined whether progerin contributes to the aging process by studying telomere function in normal and HGPS fibroblasts. We found that normal human fibroblasts undergo progressive telomere damage during cellular aging, which contributes to activating progerin production (12). This telomere damage also leads to changes in the alternative splicing in other genes. We did not observe progerin production in the absence of telomere shortening. Thus, there appears to be a synergistic relationship between telomere shortening, progerin production, and the aging process.

SUMMARY

HGPS is a rare, premature aging syndrome caused by a spontaneous, point mutation in exon 11 of the LMNA gene. Children die of heart attack or stroke in their early teens due to the progressive loss of arterial vascular smooth muscle cells and subsequent fibrosis. Farnesyltransferase inhibitor treatment of HGPS fibroblasts and mice restores nuclear and vessel architecture, respectively, and these FTIs are being investigated in a clinical trial in HGPS. Finally, progerin is activated senescence and is associated with telomere shortening, suggesting a broad role for progerin in the normal aging process.

DISCUSSION

Benz, Boston: Thank you very much Betsy. While we are lining up for questions, maybe I could ask you one. You said progerin is expressed at a relatively low frequency. I didn't know if that was low frequency of patients or low frequency of cells, but one possible explanation for that might be that there are other polymorphisms in the vicinity of the mutation or other mutations and accessory splicing factors that could account for why some people are making progerin.

Nabel, Boston: The slide showed the percentage of progerin-positive cells within a coronary artery disease plaque using an antibody that is specific for human progerin. Undoubtedly, there are other alterations in the lamin A gene that lead to activation of the splicing site.

Alexander, Atlanta: Betsy, that was absolutely terrific; and thank you for sharing this elegant research. I was very interested in your data and comments about senescence syndrome and senescence pathogenesis. You mentioned p53, which has recently been linked in interesting ways to pathways that control mitochondrial biogenesis and dysfunction. In either the patients or in your models, do they have mitochondrial dysfunction as a part of the oxidative stress?

Nabel, Boston: We've not looked specifically at mitochondrial function in the children with progeria. It's a very good question. We simply haven't looked.

Rosenblatt, Whitehouse Station: Betsy, this was a beautiful talk, and just wonderful work. I also wondered about other aging pathways and whether they intersect this, and is there any evidence that the sirulins or the Sir enzymes, Sir2 or Sir1, actually wind up impacting this pathway?

Nabel, Boston: That's an important question. We've not looked at that but, I know that there are other investigators in the field who are. More to follow.

Thibault, New York: Betsy, what a fascinating story. So, can you share with us the rationale for statins?

Nabel, Boston: So, as you know, statins alter not only farnesylation but also geranylgeranylation: a form of prenylation, which is a post-translational modification of proteins that involves the attachment of one or two 20-carbon lipophilic geranylgeranyl isoprene units from geranylgeranyl diphosphate to one or two cysteine residue(s) at the C terminus of specific proteins. Prenylation (including geranylgeranylation) is thought to function, at least in part, as a membrane anchor for proteins as well. Other investigators have hypothesized that geranylgeranylation may be part of the biochemical pathway, and so, that is the rationale. The children have normal lipid levels, including low density lipoprotein cholesterol. So, there is no reason to use statins for lipid lowering.