Page 76 - Biennial Report 2018-20 Jun 2021

thickness skin wounds), encompassing the different healing stages. Methods used were
histological and ultrastructural (high resolution electron microscopy).

Wound healing kinetics has been investigated in pigmented vs. white skin at histological,
ultrastructural and molecular levels. Full-thickness wounds were made on black and white skin
of guinea pigs, and the healing process was followed up at various time points post wound, which
coincided with the 3 main phases of healing- namely inflammation (day 9-11), proliferation (day
14-18), and remodeling (day 18 onwards). Detailed analysis of healed tissue was done to
compare if there was any difference in rate of healing, or the type of cells recruited in healing
skin in black vs white skin. ROS levels and proliferation potential of primary fibroblast cultures
derived from the black and white skin harvested from different time points post wound was
analyzed using FACS based assays. Their potential to influence keratinocyte growth was also
tested using conditioned media experiments.

Wound healing kinetics encompassing the four main phases of healing has been investigated in
detail in guinea pigs at six to nine time points encompassing the three main phases of wound
healing- namely inflammation, proliferation and remodeling.

TYPE 2 DIABETES INDUCED IMPAIRED SKIN HEALTH

In previous reports, it was reported that miR-98-5p, by targeting PPP1R15B induces ER stress
and apoptosis in cells accompanied by decreased proliferation. Malabika Datta’s group
investigated this miRNA target relationship in greater detail in the context of diabetic skin health.
miRNA hsa-miR-98-5p decreased PPP1R15B levels at the protein and transcript levels. BIP and
Chop levels together with p-eIF2a increased in the presence of miR-98-5p. miR-98-5p also
increased the levels of Bax and down-regulated the levels of Bcl-2 suggesting that this miRNA
might possibly induce apoptosis in these cells. Apoptosis as measured by annexin positive cells
was around two-fold greater in miR-98-5p transfected cells. Human fibroblasts and keratinocytes
were cultured in wells containing inserts with fibroblasts grown in the upper well insert and
induced with high glucose (25 mM) and p-AKT levels that are a marker of IGF-1 signaling were
assessed in the keratinocytes. There was a significant increase in p-Akt levels in keratinocytes
suggesting that glucose induced fibroblasts secrete substances that induce Akt phosphorylation
in HaCaT cells. 12 diabetic patients were recruited for the study so far. Analysis of stained
sections of diabetic skin revealed significant epidermal hyperplasia (thickened epidermis) and
the dermis region presented with ‘mild inflammation’ with presence of inflammatory cells as
compared to the skin of age matched healthy controls. There was increased angiogenesis in the
diabetic skin. Masson Fontana staining was performed to assess melanin content and Masson
trichrome staining for collagen content. Ultrastructural studies for diabetic and healthy control
skin samples (age and site matched) were done.

The miR-98-5p-PPP1R15B interaction was finally validated in primary keratinocytes. miRNA
mimic hsa-miR-98-5p was transfected in primary keratinocyte cells and after 48 hours RNA was
extracted, and cDNA was synthesized using 1ug of RNA. Real-time PCR for PPP1R15B and 18S
rRNA was performed. As in the HaCaT cells, a decrease in the transcript levels of PPP1R15B gene
was also observed using RT-PCR in primary keratinocytes suggesting that the miRNA significantly
decreased the mRNA levels of PPP1R15B. Moreover, there was an increase in the levels of BIP at
transcript level. As part of the second objective, WS1 (human dermal fibroblasts) cells were
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